Methods of modulation of the immune system

ABSTRACT

Manipulation of the EphB6 receptor and its active Eph partners allow for regulation of T cell responses, including TCR signalling, T cell proliferation, and induction of T cell death. Methods of modulating EphB6 are described as well as various therapeutic applications.

FIELD OF THE INVENTION

[0001] The invention relates to the field of immunology and is concernedwith protein tyrosine kinases and, more particularly, to Eph-relatedreceptor tyrosine kinases, specifically the EphB6 receptor, and itsactive partners, and methods of its manipulation for the modulation ofcellular processes.

BACKGROUND OF THE INVENTION

[0002] The regulation of development and cell proliferation in higherorganisms involves signaling through receptor tyrosine kinases (RTK).Ligand binding to the extracellular domain of RTKs induces receptordimerization or oligomerization and stimulates their intrinsic tyrosinekinase activity (Honegger et al. (1990); Kashles et al. (1991); Ueno etal. (1991); Yarden and Schlessinger (1987); Yarden and Schlessinger(1987a)). As a consequence, RTKs undergo autophosphorylation, causingfurther changes in receptor configuration and providing specific dockingsites for cytoplasmic signaling proteins containing Src-homology 2 (SH2)or phosphotyrosine binding (PTB) domains (Kavanaugh et al. (1995); Kochet al. (1991); Songyang et al. (1993)).

[0003] RTKs are divided into families on the basis of their structuralorganization (van der Geer et al. (1994)), Eph receptors forming thelargest known family, with at least 14 members (Pasquale (1997); Zhou(1998); Zisch and Pasquale (1997)). Ephs bind a group of ligands knownas ephrins (Eph family receptor interacting), eight of which arecurrently known, all membrane anchored either byglycosylphosphatidylinositol (GPI) (ephrinA1-A5), or a trans-membranedomain (ephrinB1-B3) (Drescher (1997); Pasquale (1997)). Eph receptorsare divided into two groups based upon their ligand bindingcharacteristics, EphA or EphB, according to the class of ephrin bound(Brambilla et al. (1995); Ciossek and Ullrich (1997); Gale et al.(1996); Kozlosky et al. (1995); Park and Sanchez (1997)); althoughreceptor-ligand specificity is degenerate within a group (Zhou (1998)).It is a characteristic of the Eph receptor family that their ligandsmust be membrane bound in order to be active (Davis et al. (1994);Sakano et al. (1996); Winslow et al. (1995)). This absolute requirementfor membrane anchorage of the ligand makes the formation of cell-cellcontact an obligatory event in activation of the Eph receptors.Consequently, activated receptors are concentrated in areas of cell-cellcontact.

[0004] The Eph receptors and their ligands are typically most highlyexpressed in neural and endothelial cells (Zhou (1998)) and mostdescriptions of their function concern the development of the nervoussystem and angiogenesis (Drescher et al. (1995); Friedman et al. (1996);Hornberger et al. (1999); Gao et al. (1999); Ciossek et al. (1998);Daniel et al. (1996); O'Leary et al. (1999); Pandey et al. (1995); Adamset al. (1999); Wang. et al. (1998); Yue et al. (1999)). Upon theformation of cell-cell contact, Eph receptor signaling results inreorganization of the actin cytoskeleton and integrin activation (Beckeret al. (2000); Miao et al. (2000); Zou et al. (1999); Holland et al.(1997); Huynh-Do et al. (1999)). As a result, Eph receptors generateadhesive or repulsive signals and in the neural system can guide themovement of axonal growth cones, cell migration and synapse formation(Drescher et al. (1995); Hornberger et al. (1999); Ciossek et al.(1998); Yue et al. (1999); Bohme et al. (1996); Flanagan et al. (1998);Hsueh et al. (1998); Krull et al. (1997); Monschau et al. (1997);Nakamoto et al. (1996); Mellitzer et al. (1999); Smith et al. (1997); Xuet al. (1999); Torres et al. (1998)).

[0005] The most recently identified member of the Eph family is theorphan EphB6 receptor, with a structure typical of the EphB subfamily(Gurniak et al. (1996); Matsuoka et al. (1997)). While structuralanalysis of EphB6 reveals conservation of the major EphB receptorautophosphorylation sites (Y638 and Y644), there are several criticalalterations in the tyrosine kinase domain. These include substitution ofa crucial lysine residue in the ATP binding site, resulting in areceptor that does not demonstrate detectable kinase activity (Gurniaket al. (1996); Matsuoka et al. (1997)). This casts doubt upon theability of EphB6 to undergo tyrosine phosphorylation upon ligandstimulation and thus to initiate signaling cascades in the cytoplasm.However, analogy with ErbB-3, a well-characterized catalyticallyinactive member of the EGF receptor family, suggests that EphB6 may formhetero-oligiomers with catalytically active family members. Andsimilarly, as a result of trans-phosphorylation by these activereceptors, EphB6 may recruit cytoplasmic signal transducing molecules.

[0006] Unlike other receptor tyrosine kinases, EphB6 is predominantlyexpressed in the thymus (Gurniak et al. (1996)), suggesting that it mayplay an important role in T cell differentiation. Current evidencesuggest that Eph receptors may directly interact with the TCR (T cellreceptor) signaling pathway. Eph receptors can regulate integrinactivation and cytoskeletal rearrangement (Becker et al. (2000); Miao etal. (2000); Zou et al. (1999); Holland et al. (1997); Huynh-Do et al.(1999)), both crucial events in TCR induced responses (Holsinger et al.(1998); Abraham et al. (1999); Bleijs et al. (1999); (Ticchioni et al.(1993); Valitutti et al. (1995); Wulfing et al. (1998); Wulfing et al.(1998); Snapper et al. (1998); Viola et al. (1999); Vivinus-Nebot et al.(1999)). Moreover, several Eph receptors also bind the T cell kinase Fyn(Choi et al. (1999); Ellis et al. (1996)). Indeed, high levels of EphB6expression have been detected in a population of human peripheral Tlymphocytes, but not in B cells (Shimoyama et al. (2000)). Despite itslack of kinase activity, ephrin-B1-stimulated EphB6 undergoes typrosinephosphorylation, which is provided by a catalytically active member ofthe EphB subfamily. This initiates its downstream signaling. The JunN-terminal kinase (JNK) cascade (Becker et al. (2000)) is the majorpathway downstream of the Eph receptor family, and is one of the keyregulators of T cell apoptosis (Sabapathy et al. (1999); Baker et al.(1998)). It is currently not clear whether the Eph receptor famly or anymembers, including the EphB6 receptor, have a role in such apoptosis.Regulation of this aspect of the immune system continues to bedesirable.

SUMMARY OF THE INVENTION

[0007] The present inventors have demonstrated that manipulation of thekinase-inactive EphB6 receptor and its active partners allows forregulation of T cell responses preferably cell signalling and T cellproliferation.

[0008] Further, the present inventors have determined that despite itslack of kinase activity, stimulated EphB6 undergoes tyrosinephosphorylation, and that modulation of EphB6 provides a method formodulating apoptosis, preferably for the induction of Activation inducedCell Death (AICD).

[0009] Accordingly, in its broad aspect the present invention provides amethod of modulating the immune system of an animal comprisingadministering to the animal an effective amount of a substance thatmodulates the expression, or activity of EphB6, or its active partnerthereby modulating the immune system.

[0010] In another aspect of the present invention there is provided amethod of modulating of a cell comprising administering to the cell, aneffective amount of a substance that modulates the expression, oractivity of EphB6, or its active partner thereby modulating theapoptosis.

[0011] According to one embedment of the methods of the invention thesubstances which may be used to modulate are preferably ephrin-B1, anoligomeric or monomeric soluble EphB6 receptor, a soluble EphB6 ligand,ephrin-B2, an antibody capable of binding EphB6, an antibody fragmentwhich is agonistic or antagonistic to EphB6, a physiological orsynthetic EphB6 ligand, a soluble active EphB6 partner, an antibody orfragments thereof to an EphB6 active partner, an antisense molecule toEphB6 or its active partners, or a physiological or synthetic ligand foran EphB6 active partner, more preferably the substance is Ephrin-B1 orEphrin B2.

[0012] According to another embodiment of the present invention there isprovided a method of modulating cell proliferation comprisingadministering to the cell an effective amount of a substance whichmodulates the expression or activity of an EphB6 receptor or its activepartners. Preferably the substance is ephrin-B1, an oligomeric ormonomeric soluble EphB6 receptor, a soluble EphB6 ligand, ephrin-B2, anantibody capable of binding EphB6, an antibody fragment which isagonistic or antagonistic to EphB6, a physiological or synthetic EphB6ligand, a soluble active EphB6 partner, an antibody or fragments thereofto an EphB6 active partner, an antisense molecule to EphB6 or its activepartners, or a physiological or synthetic ligand for an EphB6 activepartner.

[0013] According to yet another embodiment of the present inventionthere is provided a method of modulating a T cell response in an animalcomprising administering to the animal an effective amount of asubstance that modulates EphB6 expression or activity or that of itspartner such that the T cell response is modulated. Preferably thesubstance is ephrin-B1, an oligomeric or monomeric soluble EphB6receptor, a soluble EphB6 ligand, ephrin-B2, an antibody capable ofbinding EphB6, an antibody fragment which is agonistic or antagonisticto EphB6, a physiological or synthetic EphB6 ligand, a soluble activeEphB6 partner, an antibody or fragments thereof to an EphB6 activepartner, an antisense molecule to EphB6 or its active partners, or aphysiological or synthetic ligand for an EphB6 active partner, morepreferably the substance is Ephrin-B1 or Ephrin B2.

[0014] According to other embodiments of the methods of the presentinvention a substance which stimulates EphB6 is co-administered,preferably the substance is ephrin B1, ephrin B2., or a catalyticallyactive member of the EphB subfamily, more preferably the catalyticallyactive member of the EphB subfamily is EphB1.

[0015] According to another embodiment of the present invention there isprovided a method of treating a disorder of T-cell proliferation, anautoimmune disorder, a cell-associated autoimmune disorder, an allergicdisorder in an animal, or a host versus transplant reaction comprisingadministering to the animal an effective amount of a combination ofinhibitory or stimulatory soluble EphB6 ligand and/or soluble EphB6receptor, or a ligand to an EphB6 active partner or soluble partner,thereby treating the disorder. Preferably the substance is ephrin-B1, anoligomeric or monomeric soluble EphB6 receptor, a soluble EphB6 ligand,ephrin-B2, an antibody capable of binding EphB6, an antibody fragmentwhich is agonistic or antagonistic to EphB6, a physiological orsynthetic EphB6 ligand, a soluble active EphB6 partner, an antibody orfragments thereof to an EphB6 active partner, an antisense molecule toEphB6 or its active partners, or a physiological or synthetic ligand foran EphB6 active partner. According to a preferred embodiment thecell-associated autoimmunity is multiple sclerosis, lupus, arthritis,thyroiditis, diabetes, psoriasis, Crohn's disease or colitis. Accordingto another preferred embodiment of the method of the present invention,the allergic disorder is asthma, hyper-IgE syndrome, eosinophilicsyndrome, or a T-cell dependent graft-verus-host disease.

[0016] According to another embodiment of the present invention there isprovided a method of promoting an anti-viral immune response in ananimal comprising administering to the animal an effective amount of asubstance that modulates the expression or activity of EphB6 or itsactive Eph partner thereby promoting the antiviral response in theanimal. Preferably the substance is ephrin-B1, an oligomeric ormonomeric soluble EphB6 receptor, a soluble EphB6 ligand, ephrin-B2, anantibody capable of binding EphB6, an antibody fragment which isagonistic or antagonistic to EphB6, a physiological or synthetic EphB6ligand, a soluble active EphB6 partner, an antibody or fragments thereofto an EphB6; active partner, an antisense molecule to EphB6 or itsactive partners, or a physiological or synthetic ligand for an EphB6active partner, more preferably the substance is soluble stimulatory orinhibitory ephrin and/or a soluble EphB6 receptor.

[0017] According to another aspect of the methods of the presentinvention the animal which is subject of the methods is a mammal,preferably human.

[0018] According to another aspect of the present invention there isprovided a method for identifying a substance which is capable ofbinding to a purified and isolated EphB6 protein, comprising reactingthe protein with at least one substance which potentially can bind withthe protein under conditions which permit the formation of complexesbetween the substance and the protein, and assaying for complexes, forfree substance, for non-complexed protein, or for activation of theprotein.

[0019] According to yet another aspect of the present invention there isprovided a method for assaying a medium for the presence of an agonistor antagonist of the interaction of a purified and isolated a EphB6protein and a substance which binds to the protein which comprisesreacting the protein with a substance which is capable of binding to theprotein and a suspected agonist or antagonist substance under conditionswhich permit the formation of complexes between the substance and theprotein, and assaying for complexes, for free substance, fornon-complexed protein, or for activation of the protein.

[0020] Other features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention will now be described in relation to the drawingsin which:

[0022]FIG. 1A is a photograph of an immunoblot illustrating EphB6-Mphosphorylation in COS-7 cells upon Ephrin B1 stimulation.

[0023]FIG. 1B is a photograph of an immunoblot illustrating EphB6-Mphosphorylation in Hek-295 and NIH 3T3 cells upon Ephrin B1 stimulation.

[0024]FIG. 1C is an immunoblot illustrating time dependentphosphorylation of EphB6.

[0025]FIG. 1D is an immunoblot illustrating the effects of varyingligand concentration on phosphorylation of EphB6.

[0026]FIG. 1E is an immunoblot illustrating the effect of soluble EphB6receptor on ephrin-B1 induced EphB6 phosphorylation.

[0027]FIG. 2A is an immunoblot illustrating the phosphorylation ofEphB6-M co-expression of EphB1 in COS-7 cells.

[0028]FIG. 2B is an immunoblot illustrating the phosphorylation oftransfected EphB6-M co-transfected with EphB1 or T-7 tagged kinaseinactive EphB1 (B1-KD).

[0029]FIG. 2C is an immunoblot illustrating the ligand dependentphosphorylation of EphB6-M.

[0030]FIG. 2D is an immunoblot illustrating the induction of EphB6trans-phosphorylation by truncated EphB1.

[0031]FIG. 2E is another view of the immunoblot of FIG. 2D illustratingthe induction of phosphorylation of EphB6 by truncated EphB1.

[0032]FIG. 3 is an agarose gel illustrating the expression of EpbA2,EphB1, EphB2 and EphB6 receptors in human thymocytes and T cells.

[0033]FIG. 4A is an immunoblot illustrating pp115 co-precipitates withEphB6 in human thymocytes.

[0034]FIG. 4B is an immunoblot illustrating the time course of pp115association with EphB6.

[0035]FIG. 4C is an immunoblot illustrating pp115 as having the sameelectrophoretic mobility as c-Cb1.

[0036]FIG. 4D is an immunoblot illustrating EphB6 association with Cb1in samples immunoprecipitated from thymocyte lysates.

[0037]FIG. 4E is an immunoblot illustrating EphB6 association with Cb1in transfected cells.

[0038]FIG. 4F is an immunoblot illustrating that the G306Eloss-of-function Cb1 mutant does not bind EphB6.

[0039]FIG. 5A is an immunoblot illustrating EphB6 mediateddownregulation of Zap-70.

[0040]FIG. 5B is an immunoblot illustrating that phosphorylation ofY493F Zap-70 is not altered by EphB6.

[0041]FIG. 5C is an immunoblot illustrating the stable expression ofEphB6-M in transfected Jurkat.

[0042]FIG. 5D is an immunoblot illustrating the phosphorylation ofEphB6-M in Jurkat with stimulation by ephrin-B1.

[0043]FIG. 5E is an immunoblot illustrating EphB6 downregulation of thephosphorylation of Zap-70.

[0044]FIG. 5F is an immunoblot illustrating EphB6 downregulation of thephosphorylation of Zap-70 associated CD3ζ in Jurkat.

[0045]FIG. 6A is an immunoblot illustrating the effect of ephrin-B1 onTCR induced activation of Lck.

[0046]FIG. 6B is an immunoblot illustrating the effect of EphB6 on TCRinduced activation of Lck.

[0047]FIG. 7A provides a series of graphs illustrating the effect ofephrin-B1 on TCR mediated upregulation of CD25.

[0048]FIG. 7B provides a series of graphs illustrating the effect ofoverexpression of EphB6 on TCR mediated upregulation of CD25.

[0049]FIG. 8A is a histogram illustrating the enhancement of CD25upregulation by dominant negative EphB6.

[0050]FIG. 8B is a histogram illustrating the enhancement of CD25upregulation by dominant negative EphB6.

[0051]FIG. 8C is a further histogram illustrating the inhibition of CD25regulation.

[0052]FIG. 9A is an immunoblot illustrating stable expression of EphB6receptor in the mature T cell line Jurkat.

[0053]FIG. 9B is a series of figures illustrating the effect ofoverexpression of EphB6 upon induction of apoptosis in T-cells.

[0054]FIG. 10A is a histogram illustrating the EphB6-dependent increasein expression of TNFα.

[0055]FIG. 11A is a histogram illustrating that the ephrin-B1 inhibitsexpression of TNFR II, but not TNFR I.

[0056]FIG. 11B is a histogram illustrating that the overexpressed EphB6receptor inhibits expression of TNFR II, but not TNFR I.

[0057]FIG. 12 are immunoblots illustrating that EphB6 receptor is ableto prevent activation of p54 JNK.

[0058]FIG. 13 provides an illustration of a model of EphB6 receptorinteraction with the TCR signaling pathway.

DETAILED DESCRIPTION OF THE INVENTION

[0059] As stated above, the present inventors have demonstrated thatmodulation of the kinase-inactive EphB6 receptor allows for modulationof the immune system.

[0060] In particular, the inventors have determined that despite itslack of kinase activity, the EphB6 undergoes tyrosine phosphorylationupon stimulation with a substance, preferably membrane-bound or solubleoligomerized ephrin-B1. They have also demonstrated that EphB6 can betrans-phosphorylated by catalytically active members of the EphBsubfamily, in particular, by EphB1.

[0061] The present inventors also demonstrate that the EphB6 receptorassociates with c-Cb1, a protein central to the regulation of TCRsignaling. Cb1 binding to EphB6 is constitutive, but is lost uponintroduction of a Cb1 G306E ‘loss of function’ mutation In contrast,oncogenic 70-Z Cb1 binds EphB6 essentially like wild type Cb1.

[0062] The present inventors have also determined that, overexpressionof the EphB6 receptor in T cells resulted in inhibition of anti-CD3dependent phosphorylation of the TCR-associated kinase Zap-70 and itsassociated CD3ζ chain. This appeared to be mediated by a primaryinhibition of the activity of the src-like kinase Lck. Ultimately, thisblockage in TCR signaling results in a failure of T cell response,inhibiting upregulation of CD25 expression.

[0063] Stable overexpression of the EphB6 receptor was also found tosignificantly enhance TCR-mediated apoptosis in an ephrin-B1-dependentmanner, thus demonstrating that modulation of EphB6 provides a methodfor regulating the induction of AICD.

[0064] As used herein “Behaviour of cells of the immune system” meansthe sum of the ability of cells to respond to a given stimulus and tointeract with their environment, in particular, the rate at which theyundergo proliferation, differentiation and cell death and develop immuneresponses.

[0065] As used herein “in conjunction with” or “co-adminstration” meansconcurrently, before or following adminstration of a first substance.

[0066] As used herein “animal” means any member of the animal kingdom,including, preferably, humans.

[0067] As used herein, administration of an “effective amount” of asubstance or compound(s) of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. The effective amount of a compound of theinvention may vary according to factors such as the disease state, age,sex, and weight of the animal. Dosage regima may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0068] The term “active partner” as used herein means any EphB6interacting tyrosine kinase receptor, preferably a member of the Ephfamily of tyrosine receptor kinases.

[0069] The EphB6 Receptor

[0070] The standard features of the EphB6 receptor place it in the EphBsubfamily (Gurniak et al. (1996); Matsuoka et al. (1997)). EphBreceptors are stimulated by membrane bound ephrin-B ligandsdemonstrating highly degenerate specificity, with ephrin-B1 andephrin-B2 activating most EphB receptors (Zhou et al. (1998)).

[0071] The murine EphB6 receptor was reported to be expressedpredominantly in thymocytes (Gurniak et al. (1996)), suggesting that itmay have an important role in T cell differentiation. By RT-PCR, thepresent inventors detected EphB6 expression in both human thymocytes andmature peripheral blood T cells, as well as in the T cell line Jurkat(FIG. 3). Two catalytically active members of the Eph family, EphB1 andEphB2, were also expressed throughout the T cell lineage, while theEphA2 receptor could only be detected in thymocytes. The persistentexpression of EphB6 across the T cell lineage suggested it might notonly be important during differentiation, but also in mature T cellfunction.

[0072] Due to the membrane bound nature of both the Eph receptor andephrin ligand, an important feature of receptor-ligand interaction isthe necessity for the formation of cell-cell contact. As activation ofthe TCR complex occurs in an area of T-cell contact with anantigen-presenting cell, activated TCR complexes may potentially bebrought into close proximity with EphB receptors. TCR signalingresponses are dependent upon re-organization of the actin cytoskeletonand signals transmitted via integrin receptors, both processes regulatedby activated Eph receptors in a variety of cells (Holsinger et al.(1998); Abraham et al. (1999); Bleijs et al. (1999); Ticchioni et al.(1993); Valitutti et al. (1995); Wulfing and Davies (1998); Wulfing etal. (1998); Snapper et al. (1998); Viola et al. (1999); Vivinus-Nebot etal. (1999)). The potential for productive interaction between these tworeceptor pathways therefore appeared high.

[0073] The inventors demonstrate that activation of the T cell receptorresults in association of phosphorylated carol protein with the EphB6receptor. The ability of Cb1 to bind EphB6 suggests that analogous tothe EGF receptor, EphB6 expression may be regulated by Cb1 mediatedmodification. It is now clear that Cb1 is responsible for the physicaldownregulation of many receptors through induction of receptorubiquitination (Levkowitz et al. (1998), Wang et al. (1999), Lee et al(1999), Miyake et al. (1999). Addition of ubiquitin moieties to thelysine residues of a protein targets it for degradation (Hershko et al.(1998), either in cytoplasmic proteasomes or in lysosomes. Cb1 bindinginduces ubiquitination of the EGF, ErbB1, PDGF and CSF receptors, anability derived from its ring finger domain (mutated in 70-Z Cb1). Thering-finger domain appears to be an E3 ubiquitin-ligase (Joazeiro et al.(1999)), responsible for the transfer of ubiquitin from a carrierprotein (E2) to the target, thus controlling the specificity ofdegradation. As all receptors known to bind Cb1 undergo ubiquitination,it is likely that EphB6 function will be similarly regulated. This mayprovide a potential mechanism for regulating the effective cell surfaceexpression level of the EphB6 receptor.

[0074] The inventors also demonstrate that overexpression or activationof the EphB6 receptor in T-cells can modulate signaling through theT-cell antigen receptor. Overexpression of the EphB6 receptor results inan inhibition of anti-CD3 induced activation of the Src-family kinaselck and subsequently phosphorylation of Zap-70 kinase and its associatedCD3ζ chain.

[0075] Stimulation of the TCR leads in particular to induction of bothIL-2 production and CD25 (IL-2Rα) expression; thus potentially inducingexpansion of activated T-cell populations (Chambers et al. (1997)). TCRmediated induction of CD25 requires activation of the TCR-associatedkianses. The investigators demonstrate that supression of the earlyevents of TCR signaling by EphB6 ultimately translates into aninhibition of T-cell response, in particular, CD25 upregulation. Inagreement, Ephrin-B1 stimulation of thymocytes, which naturally expresshigh levels of EphB6, prevents TCR mediated upregulation of IL-2receptor expression. The inhibitory effect of the endogenous EphB6receptor upon the TCR complex was confirmed by the ability of a dominantnegative form of EphB6 to enhance the TCR-induced upregulation of CD25in T-cells. T lymphocyte homeostasis is precisely regulated, with GusTCR co-stimulatory events required for finely tuned control of cellfate, these signals regulating both proliferative and apoptic pathways(Chambers et al. (1997), Janeway et al. (1994)). Clearly EphB6 acts asan effective TCR co-receptor, influencing the response of cells to TCRstimulation.

[0076] Expression of CD25 is central to the IL-2 driven clonal expansionthat occurs upon exposure of mature T-cells to antigen. Failure toexpress the high affinity IL-2R complex composed of the α (CD25), β andγ chains prevents the development of the necessary IL-2 autocrineproliferative loop (Nelson and Willerford, 1998). Thus, while notwishing to be bound by any one theory, one of the biological functionsof EphB6, in conjunction with other EphB receptors, may be to controlthe clonal expansion of antigen activated T-cells through suppression ofantigen-induced CD25 expression and associated events. Severalalternative models of EphB6 function also become apparent. Underphysiological conditions, stimulation of the EphB6 receptor may serve tomaintain activation of the TCR signaling pathway below a certainthreshold, preventing premature activation by inappropriate low affinityTCR interactions. Or alternatively, the presence of varying ephrin-Bligands may modify the ability of T-cells to respond to antigenspresented on different cell-surfaces. Failure to correctly regulate TCRsignaling may lead to uncontrolled activation or undesirable activationupon very low affinity interaction with antigen. The consequences ofthese events may be multiple but include autoimmune reactions, as lowaffinity self-self interactions are not properly regulated, orrecognition by the T-cell of inappropriate target cells due to theabsence of appropriate targeting by Eph receptor engagement, orinadvertent activation of bystander cells due to cytokine overproductionby uncontrolled activated cells.

[0077] Modulation of EphB6

[0078] Antibodies

[0079] Antibodies represent a class of substances that may be usedadvantageously to modulate the activity of the EphB6 receptor.Antibodies may be used to either inhibit, or stimulate the EphB6receptor. Antibodies can be prepared which bind a distinct epitope in anunconserved region of the protein. An unconsumed region of the proteinis one that does not have substantial sequence homology to otherproteins.

[0080] Conventional methods can be used to prepare the antibodies. Forexample, by using a peptide of the EphB6 receptor, polyclonal antiseraor monoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the peptide which elicits an antibody response in the mammal.Techniques for conferring immunogenicity on a peptide includeconjugation to carriers or other techniques well known in the art. Forexample, the protein or peptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayprocedures can be used with the immunogen as antigen to assess thelevels of antibodies. Following immunization, antisera can be obtainedand, if desired, polyclonal antibodies isolated from the sera.

[0081] To produce monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from an immunized animal and fused withmyeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g., the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256, 495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al. Monoclonal Antibodiesin Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), andscreening of combinatorial antibody libraries (Huse et al., Science 246,1275 (1989)). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with the peptide and themonoclonal antibodies can be isolated. Therefore, the invention alsocontemplates hybridoma cells secreting monoclonal antibodies withspecificity for the EphB6 receptor as described herein.

[0082] The term “antibody” as used herein is intended to includefragments thereof which also specifically react with an EphB6 receptor,or peptide thereof, having the activity of the EphB6 receptor.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above.For example, F(ab′)2 fragments can be generated by treating antibodywith pepsin. The resulting F(ab′)2 fragment can be treated to reducedisulfide bridges to produce Fab′ fragments.

[0083] Chimeric antibody derivatives, i.e., antibody molecules thatcombine a non-human animal variable region and a human constant regionare also contemplated within the scope of the invention. Chimericantibody molecules can include, for example, the antigen binding domainfrom an antibody of a mouse, rat, or other species, with human constantregions. Conventional methods may be used to make chimeric antibodiescontaining the immunoglobulin variable region which recognizes the geneproduct of EphB6 antigens of the invention (See, for example, Morrisonet al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al.,Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP171496; European Patent Publication 0173494, UnitedKingdom patent GB 2177096B). It is expected that chimeric antibodieswould be less immunogenic in a human subject than the correspondingnon-chimeric antibody.

[0084] Monoclonal or chimeric antibodies specifically reactive with aprotein of the invention as described herein can be further humanized byproducing human constant region chimeras, in which parts of the variableregions, particularly the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such immunoglobulin molecules may bemade by techniques known in the art, (e.g., Teng et al., Proc. Natl.Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., ImmunologyToday, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)),and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies canalso be commercially produced (Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.)

[0085] Specific antibodies, or antibody fragments, reactive againstEphB6 receptor proteins may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with peptides produced from the nucleic acid molecules ofthe EphB6 receptor. For example, complete Fab fragments, VH regions andFV regions can be expressed in bacteria using phage expression libraries(See for example Ward et al., Nature 341, 544-546: (1989); Huse et al.,Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554(1990)). Alternatively, a SCID-hu mouse, for example the model developedby Genpharm, can be used to produce antibodies or fragments thereof.

[0086] Antibodies specifically reactive with the EphB6 receptor, orderivatives thereof, such as enzyme conjugates or labeled derivatives,may be used to detect the EphB6 receptor in various biologicalmaterials, for example they may be used in any known immunoassays whichrely on the binding interaction between an antigenic determinant of theEphB6 receptor, and the antibodies. Examples of such assays areradioimmunoassays, enzyme immunoassays (e.g. ELISA),immunoflurorescence, immunoprecipitation, latex agglutination,hemagglutination and histochemical tests. Thus, the antibodies may beused to detect and quantify the EphB6 receptor in a sample in order todetermine its role in particular cellular events or pathological states,and to diagnose and treat such pathological states.

[0087] In particular, the antibodies of the invention may be used inimmuno-histochemical analyses, for example, at the cellular andsub-subcellular level, to detect the EphB6 receptor, to localise it toparticular cells and tissues and to specific subcellular locations, andto quantitate the level of expression.

[0088] Cytochemical techniques known in the art for localizing antigensusing light and electron microscopy may be used to detect the EphB6receptor. Generally, an antibody of the invention may be labelled with adetectable substance and the EphB6 receptor may be localised in tissuebased upon the presence of the detectable substance. Examples ofdetectable substances include various enzymes, fluorescent materials,luminescent materials and radioactive materials. Examples of suitableenzymes include horseradish peroxidase, biotin, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material includeradioactive iodine I-125, I-131 or 3-H. Antibodies may also be coupledto electron dense substances, such as ferritin or colloidal gold, whichare readily visualised by electron microscopy.

[0089] Indirect methods may also be employed in which the primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against the EphB6receptor. By way of example, if the antibody having specificity againstthe EphB6 receptor is a rabbit IgG antibody, the second antibody may begoat anti-rabbit gamma-globulin labelled with a detectable substance asdescribed herein.

[0090] Where a radioactive label is used as a detectable substance, theEphB6 receptor may be localized by autoradiography. The results ofautoradiography may be quantitated by determining the density ofparticles in the autoradiographs by various optical methods, or bycounting the grains.

[0091] Soluble proteins represent another class of substances that maybe used advantageously to modulate the activity of the EphB6 receptor.Soluble proteins can be prepared by a number of conventionalmethodologies. GST fusion proteins of Eph receptor and ephrinextracellular domains, or activated or inactive variants thereof, can becreated in the pGEX vector series (Pharmacia Biotech, Uppsala). When thevectors containing the cDNAs are transformed into bacteria by heat shockuptake, expression of the GSI fusion proteins can be induced with 1 mMIPTG. After growth bacteria can be lysed by sonication and the additionof mild detergents. the resulting supernatant can be clarified bycentrifugation and the released GST-fusion proteins purified by bindingto glutathione-sepharose. After extensive washing these complexes can bechecked for purity and quantitated by reference to standard proteins ofsimilar molecular weight after staining with coomassie blue.Alternatively fusions of the Eph or ephrin proteins with MBP, His,thioHis, Fc, Myc tag, HA tag, or other epitopes or domains may be usedto allow other purification procedures to be utilized which may resultin preferable activity of the purified protein.

[0092] It would also be apparent to one skilled in the art that theabove described methods may be used to study the expression of the EphB6receptor and, accordingly, will provide further insight into the role ofthe EphB6 receptor in cells.

[0093] Antisense Oligonucleotides

[0094] Antisense oligonucleotides that are complimentary to a nucleicacid sequence from the EphB6 receptor can also be used in the methods ofthe present invention to modulate the expression and/or activity EphB6receptors.

[0095] Accordingly, the present invention provides a method ofmodulating the immune system by modulating the expression and/oractivity EphB6 receptors comprising administering an effective amount ofan antisense oligonucleotide that is complimentary to a nucleic acidsequence from the EphB6 receptor to an animal in need thereof.

[0096] The term “antisense oligonucleotide” as used herein means anucleotide sequence that is complimentary to its target.

[0097] The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

[0098] The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

[0099] Other antisense oligonucleotides of the invention may containmodified phosphorous, oxygen heteroatoms in the phosphate backbone,short chain alkyl or cycloalkyl intersugar linkages or short chainheteroatomic or heterocyclic intersugar linkages. For example, theantisense oligonucleotides may contain phosphorothioates,phosphotriesters, methyl phosphonates, and phosphorodithioates. In anembodiment of the invention there are phosphorothioate bonds linksbetween the four to six 3′-terminus bases. In another embodimentphosphorothioate bonds link all the nucleotides.

[0100] The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

[0101] The antisense nucleic acid molecules may be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense nucleic acid molecules of the inventionor a fragment thereof, may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed with mRNA or the native genee.g. phosphorothioate derivatives and acridine substituted nucleotides.The antisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

[0102] The antisense oligonucleotides may be introduced into tissues orcells using techniques in the art including vectors (retroviral vectors,adenoviral vectors and DNA virus vectors) or physical techniques such asmicroinjection. The antisense oligonucleotides may be directlyadministered in vivo or may be used to transfect cells in vitro whichare then administered in vivo. In one embodiment, the antisenseoligonucleotide may be delivered to macrophages and/or endothelial cellsin a liposome formulation.

[0103] Modulation of EphB6 Promoter

[0104] As would be readily apparent to those skilled in the art, it isalso possible to modulate EphB6 through manipulation of its promoter.One or more alterations to a promoter sequence of the EphB6 may increaseor decrease promoter activity, or increase or decrease the magnitude ofthe effect of a substance able to modulate the promoter activity.

[0105] “Promoter activity” is used to refer to the ability to initiatetranscription. The level of promoter activity is quantifiable forinstance by assessment of the amount of mRNA produced by transcriptionfrom the promoter or by assessment of the amount of protein productproduced by translation of mRNA produced by transcription from thepromoter. The amount of a specific mRNA present in an expression systemmay be determined for example using specific oligonucleotides which areable to hybridise with the mRNA and which are labelled or may be used ina specific amplification reaction such as the polymerase chain reaction.

[0106] Substances which affect the EphB6 promoter's activity may also beidentified using the methods of the invention by comparing the patternand level of expression of a reporter gene, in cells in the presence,and in the absence of the substance. Accordingly a method for assayingfor the presence of an agonist or antagonist of EphB6 promoter activityis provided comprising providing a cell containing a reporter gene underthe control of the promoter with a substance which is a suspectedagonist or antagonist under conditions which permit interaction andassaying for the increase or decrease of reporter gene product.

[0107] Apoptosis of Cells

[0108] Activation induced apoptosis (programmed cell death) maintainshomeostasis and immune tolerance by regulating the number and type ofantigen stimulated T-cells in circulation. Activation induced cell death(ACID) can be provoked in antigen-stimulated T-cells to eliminatepotentially harmful cells and excessive clonotypes, thus preserving thefunctional balance of the immune system, preventing autoimmune andlympho-proliferative disorders (Park et al. (1997); Davis et al. (1994);Sakano et al. (1996)).

[0109] The investigators demonstrate that stable overexpression of theEphB6 receptor significantly enhances TCR-mediated apoptosis in anephrin-B1-dependent manner in the mature T-cell line Jurkat; a commonlyused model of pre-stimulated mature T cells in ACID studies. ActiveT-cell apoptosis is driven by the antigen-induced expression of the FASLand TNF death cytoidnes (Friedman et al. (1996); Hornberger et al.(1999); Gao et al. (1999); Ciossek et al. (1998)). The increasedapoptosis observed in EphB6 overexpressing cells appears to be due inpart to increased TNP production. Although TNF efficiently activatesboth the TNFR-I and TNFR-II receptors, studies suggest that only TNFR-Iis coupled to a caspase cascade (Kozlosky et al. (1995)) and thus may bethe predominant transmitter of the apoptic signal (Daniel et al. (1996);O'Leary et al. (1999)). Expression of TNFR-II, but not TNFR-I, issuppressed upon incubation of control and EphB6 overexpressing cellswith ephrin-B1. Although activation of the TCR overrides this effect incontrol cells, overexpression of EphB6 maintains the ephrin-B1-induceddown regulation of TNFR-II. Anti-CD3 stimulation of EphB6 overexpressingcells also reduces TNFR-II expression, while it has no effect upon thereceptor in control cells, suggesting that the basal activity ofoverexpressed EphB6 receptor is sufficient to make cells more sensitiveto the induction of apoptosis. The EphB6-induced imbalance in TNFR-I andTNFR-II expression is interestingly similar to the situation observed inthe T-cells of aging humans, where TNFR-I is constitutively expressedand TNFR-II is downregulated. These T-cells are hypersensitive toTNF-induced apoptosis, which is probably responsible for increasingT-cell deficiency in old-age (Pandey et al. (1995)). Eph receptors couldpotentially be responsible for this alteration in TNP receptorexpression and modulation of their activity could improve TNFR-IIexpression.

[0110] TNFR-I and TNFR-II employ only partially distinct signalingpathways, both initiating the n-terminal JUN kinase cascade (Kozlosky etal. (1995)). Activation of the JNK pathway is required to protect cellsfrom TNF-mediated apoptosis (Adams et al. (1999), Wang et al. (1998)).Overexpression of EphB6 strongly inhibits long-term anti-CD3 induced JNKstimulation. This effect is highly specific, several other potentiallyanti-apoptic pathways, including Akt activation and Bcl-2 expression,are not affected. The elimination of JNK-JUN signaling reportedlyenhances TNF-induced apoptosis (Adams et al. (1999); Wang et al.(1998)), suggesting that selective inhibition of the JUN kinase pathwaycould further the promotion of AICD by EphB6.

[0111] Thus, and while not wishing to be bound by any particular theory,the increase in inducible programmed cell death in EphB6 overexpressingcells can be attributed at least in part to increased TNF production,complemented by an alteration in the balance between TNFR-I and TNFR-IIexpression to favor the pro-apoptic TNFR-I. Accordingly, the presentinvention provides a method of regulating the immune system, preferablyregulating lymphocyte apoptosis, preferably AICD, by providing aneffective amount of a substance capable of modulation of EphB6 and itsactive partners, thereby modulating the immune system.

[0112] The high level of EphB6 expression in thymocytes also suggeststhat EphB6 may play an important role in vivo in the negative andpositive selection of thymocytes, regulating the induction of theapoptic pathway in cells that fail to be positively selected. Failure toproperly regulate negative selection can lead to the emergence ofauto-reactive T-cells in the periphery leading to the development ofautoimmune diseases. In the peripheral blood, failure to eliminateactivated T-cells may result in T-cell lymphoproliferative disorders orauto-immune disorders as the result of an inability to eliminate selfreactive T-cells.

[0113] Therapeutic Uses

[0114] As just discussed, the EphB6 receptor of the invention is likelyinvolved in the regulation of cell signalling pathways that control celldeath. Accordingly, the present invention provides a method ofmodulating cell death or apoptosis comprising administering to a cell oranimal in need thereof, an effective amount of a substance thatmodulates EphB6, in order to modulate the cell death. Examples ofsubstance which may be used to modulate EphB6 include antibodies,soluble EphB6, soluble ephrins, antisense nucleic acids, organicsubstances that modulate the interaction of EphB6 with ligands andactive partners either alone or in combination. The term “effectiveamount” as used herein means an amount effective, at dosages and forperiods of time necessary to achieve the desired results.

[0115] In another aspect the present invention provides a method ofmodulation of cell proliferation. In one embodiment, the inventionprovides a method of inhibiting or reducing cell proliferation, such asin neoplasia, by administering to a cell or animal an effective amountof an agent that promotes the expression or the biological activity ofthe EphB6 receptor or its active Eph partners, such that there is aninhibition or reduction in cell prolifereation.

[0116] In another embodiment, the present invention provides a method ofinducing cell proliferation by administering to a cell or an animal aneffective amount of a substance that inhibits the expression or thebiological activity of the EphB6 receptor, or blocks the phosphorylationof the said receptor or its active Eph partners, such that there is aninduction of cell proliferation. Substances that inhibit the activity ofthe EphB6 receptor include antibodies to EphB6 receptor. Substances thatinhibit the expression of the EphB6 gene include antisenseoligonucleotides to an EphB6 receptor nucleic acid sequence.

[0117] In addition to antibodies and antisense oligonucleotides, othersubstances that modulate EphB6 receptor expression or activity may alsobe identified, as well as substances that block the phosphorylation ofEphB6. Substances that affect EphB6 receptor activity can be identifiedbased on their ability to bind to the EphB6 receptor.

[0118] Substances which can bind with the EphB6 receptor of theinvention may be identified by reacting the EphB6 receptor with asubstance which potentially binds to the EphB6 receptor, and assayingfor complexes, for free substance, or for non-complexed EphB6 receptor,or for activation of the EphB6 receptor. In particular, a yeast twohybrid assay system may be used to identify proteins which interact withthe EphB6 receptor (Fields, S. and Song, O., 1989, Nature, 340:245-247)or a ligand binding or ligand replacement assay system (Blechman, J. M.et al. (1993); Blechman, J. K et al. (1995); Lev et al. (1993)). Systemsof analysis which also may be used include ELISA, BIAcore(Bartley, T.D., et al. (1994)).

[0119] A protein ligand for the Eph receptors can be isolated by usingthe extracellular domain of the receptor as an affinity reagent.Concentrated cell culture supernatants can be screened for receptorbinding activity using immobilized receptor in a surface plasmonresonance detection system (BIAcore). Supernatants from selected celllines can then be fractionated directly by receptor affinitychromatography.

[0120] Conditions which permit the formation of substance and EphB6receptor complexes may be selected having regard to factors such as thenature and amounts of the substance and the protein.

[0121] The substance-protein complex, free substance or non-complexedproteins may be isolated by conventional isolation techniques, forexample, salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof. To facilitate the assay of thecomponents, antibody against the EphB6 receptor or the substance, orlabelled EphB6 receptor, or a labelled substance may be utilized. Theantibodies, proteins, or substances may be labelled with a detectablesubstance as described above.

[0122] Substances which bind to and activate the EphB6 receptor of theinvention may be identified by assaying for phosphorylation of thetyrosine residues of the protein.

[0123] Substances which bind to and inactivate the EphB6 receptor of theinvention may be identified by assaying for reduction in phosphorylationof the protein.

[0124] The EphB6 receptor, or the substance used in the method of theinvention may be insolubilized. For example, the EphB6 receptor orsubstance may be bound to a suitable carrier. Examples of suitablecarriers are agarose, cellulose, dextran, Sephadex, Sepharose,carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin,plastic film, plastic tube, glass beads, polyamine-methylvinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleicacid copolymer, nylon, silk, etc. The carrier may be in the shape of,for example, a tube, test plate, beads, disc, sphere etc.

[0125] The insolubilized protein or substance may be prepared byreacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

[0126] The proteins or substance may also be expressed on the surface ofa cell using the methods described herein.

[0127] The invention also contemplates a method for assaying for anagonist or antagonist of the EphB6 receptor. The agonist or antagonistmay be an endogenous physiological substance or it may be a natural orsynthetic substance. Substances that are capable of binding the EphB6receptor may be identified using the methods set forth herein.

[0128] The invention also contemplates assaying for an antagonist oragonist of the EphB receptor and its active partner or partnerspreferably an Eph receptor.

[0129] It will be understood that the agonists and antagonists that canbe assayed using the methods of the invention may act an one or more ofthe binding sites an the protein or substance including agonist bindingsites, competitive antagonist binding sites, non-competitive antagonistbinding sites or allosteric sites.

[0130] The invention also makes it possible to screen for antagoniststhat inhibit the effects of an agonist of the EphB6 receptor or itsactive partners. Thus, the invention may be used to assay for asubstance that competes for the same binding site of the EphB6 receptoror its active partners.

[0131] The methods described above may be used to identify a substancewhich is capable of binding to an activated EphB6 receptor or its activepartners, and to assay for an agonist or antagonist of the binding ofactivated EphB6 receptor or its partners, with a substance which iscapable of binding with activated EphB6 receptor or its partners. Anactivated (i.e. phosphorylated) the EphB6 receptor may be prepared usingthe methods described (for example in Reedijk et al. The EMBO Journal,11(4):1365, 1992) for producing a tyrosine phosphorylated protein.

[0132] It will also be appreciated that intracellular substances whichare capable of binding to EphB6 or its active partners may be identifiedusing the methods described herein.

[0133] The invention further provides a method for assaying for asubstance that affects an EphB6 receptor regulatory pathway comprisingadministering to a human or animal or to a cell, or a tissue of ananimal, a substance suspected of affecting a EphB6 receptor regulatorypathway, and quantitating the EphB6 receptor or nucleic acids encodingthe EphB6 receptor, or examining the pattern and/or level of expressionof EphB6 receptor, in the human or animal or tissue, or cell. EphB6receptor may be quantitated and its expression may be examined using.the methods described herein.

[0134] The substances identified by the methods described herein, may beused for modulating EphB6 receptor regulatory pathways and accordinglymay be used in the treatment of conditions involving perturbation ofEphB6 receptor signaling pathways. In particular, the substances may beparticularly useful in the treatment of disorders of cell death.

[0135] As stated previously, EphB6 receptor may be involved inmodulating cell proliferation and stimulators and inhibitors of theEphB6 receptor may be useful in modulating disorders involving cellproliferation such as neoplasia and autoimmunity, such as for example,substances that stimulate the EphB6 receptor (for example, identifiedusing the methods of the invention) may be used to stimulate cell deathor apoptosis, and inhibitors could be used where an increase in T cellproliferation would be advantageous.

[0136] Peptide Mimetics

[0137] The present invention also includes peptide mimetics of the EphB6receptor of the invention. For example, a peptide derived from a bindingdomain of an EphB6 protein will interact directly or indirectly with anassociated molecule in such a way as to mimic the native binding domain.Such peptides may include competitive inhibitors, enhancers, peptidemimetics, and the like. All of these peptides as well as moleculessubstantially homologous, complementary or otherwise functionally orstructurally equivalent to these peptides may be used for purposes ofthe present invention.

[0138] “Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures which may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a pep tide, or enhancer or inhibitor of the invention. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to a peptide of the invention.

[0139] Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino add analogues may be used to constrain aminoadd residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

[0140] Peptides of the invention may also be used to identify leadsounds for drug development. The structure of the peptides describedherein can be readily determined by a number of methods such as NMR andX-ray crystallography. A comparison of the structures of peptidessimilar in sequence, but differing in the biological activities theyelicit in target molecules can provide information about thestructure-activity relationship of the target. Information obtained fromthe examination of structure-activity relationships can be used todesign either modified peptides, or other small molecules or leadcompounds which can be tested for predicted properties as related to thetarget molecule. The activity of the lead compounds can be evaluatedusing assays similar to those described herein.

[0141] Information about structure-activity relationships may also beobtained from co-crystallization studies. In these studies, a peptidewith a desired activity is crystallized in association with a targetmolecule, and the X-ray structure of the complex is determined. Thestructure can then be compared to the structure of the target moleculein its native state, and information from such a comparison maybe usedto design compounds expected to possess.

[0142] The invention also makes it possible to screen for antagoniststhat inhibit the effects of an EphB6 receptor. Thus, the invention maybe used to assay for a substance that anatagonizes or blocks the actionof the receptor.

[0143] The invention further provides a method for assaying for asubstance that affects the EphB6 receptor, comprising administering to anon-human animal or to a tissue of an animal, a substance suspected ofaffecting the activity or action of the receptor and quantitating theeffect an CD25 expression in the human animal or tissue. CD25 may bequantitated and its expression may be examined using the methodsdescribed herein.

[0144] Substances identified by the methods described herein, may beused for modulating EphB6 receptor activity or action and accordinglymay be used in the treatment of conditions involving perturbation of theprotein. In particular, the substances may be particularly useful in thetreatment of disorders of T-cell proliferation. In addition, theapplication of a proper combination of inhibitory or stimulatory solubleligand or soluble receptors should prevent T lymphocyte-target cellinteraction and decrease host reaction versus transplant, thusinhibiting transplant rejection. As well, by virtue of the methods andsubstances of the present invention, the employment of inhibitory orstimulatory soluble ligands and soluble receptors may be used fortreatment or slowing of autoimmune disorders. Such autoimmune disordersmay include cell-associated autoimmunities such as multiple sclerosis,lupus, arthritis, thyroiditis, diabetes, psoriasis and Crohn's diseaseand colitis. In addition, the methods and substances may be used totreat allergic disorders such as asthma and hyper-IgE and eosinophilicsyndromes and T-cell dependent graft-verus-host reactions. As well, byvirtue of the substances and methods described herein, solublestimulatory or inhibitory ephrins and soluble receptors could promoteboth T lymphocyte adhesion and T cell response to infected cells, thusaccelerating and increasing anti-viral immune response.

[0145] It is also envisaged that the DNA sequences of the EphB6 receptoror its active partners might be determined in order to assay forchanges, preferably disease-causing mutations that may be used asindicators of disease prognosis or as aids to inform treatment of thesediseases.

[0146] Pharmaceutical Compositions

[0147] The above described substances may be formulated intopharmaceutical compositions for adminstration to subjects in abiologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The substances may beadministered to living organisms including humans, and animals.

[0148] Administration of a therapeutically active amount ofpharmaceutical compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a substance may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the substanceto elicit a desired response in the individual. Dosage regima may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

[0149] An active substance may be administered in a convenient mannersuch as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending an the route of administration, the activesubstance may be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound. If the active substance is a nucleic acidencoding, for example, a modified EphB6 receptor it may be deliveredusing techniques known in the art.

[0150] The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985) or Handbook ofPharmaceutical Additives (compiled by Michael and Irene Ash, GowerPublishing Limited, Aldershot, England (1995)). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and may be contained in buffered solutions with asuitable pH and/or be iso-osmotic with physiological fluids. In thisregard, reference can be made to U.S. Pat. No. 5,843,456. As will alsobe appreciated by those skilled, administration of substances describedherein may be by an inactive viral carrier.

[0151] Experimental Models

[0152] The invention also provides methods for studying the function ofEphB6 and its impact on cells of the immune system.

[0153] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLES General Methods for Examples 1-5 Antibodies and RecombinantProteins

[0154] Monoclonal anti-phosphotyrosine was obtained from UpstateBiotechnology, Inc. (Lake Placid, N.Y.). Antibodies to EphB6, MYC,Zap-70 and LCK were purchased from Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.). Soluble dimerized Ephrin-B1 and soluble EphB6 receptorswere purchased from R&D Systems. Anti-human CD3 was purchased fromSerotec (UK) and anti-T7 from Novagen.

[0155] Immunoprecipitation and Western Blotting

[0156] Cells were quickly resuspended in ice cold lysis bufferconsisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%Triton X-100, 1 mM ethylene glycol-bis(β-aminoethylether)-N,N,N′-N′-tetraacetic acid (EGTA), 10 μg/mlleupeptin, 10 μg/ml aprotinin, 1 mm PMSF, 1 mM Na-orthovanadate and 50mM NaF after solubilization on ice for 15 minutes, debris was removed bycentrifugation at 12,000 g for 10 minutes at 4° C. antibodies and 20 μlof 50% protein G sepharose were added to cleared lysates and incubatedat 4° C. with constant shaking for 12-16 hours. Immunoprecipitates werecollected by a brief centrifugation and washed 3-4 times in lysis buffer(without PMSF) before addition of SDS sample buffer. Samples wereseparated on SDS-polyacrylamide gels and transferred to nitrocellulosemembranes (Amersham, Arlington Heights, Ill.). Membranes were blockedovernight at 4° C. with 7% blotting grade non-fat milk (Biorad,Richmond, Calif.) in PBS. Immunoblotting antibodies were added atoptimal dilutions in PB-ST (0.1% Tween-20) and incubated at 4° C.overnight. After extensive washing with PBS-T, bound antibodies weredetected using horseradish-peroxidase conjugated donkey anti-rabbit orsheep anti-mouse antibodies (Amersham, Arlington Heights, Ill.) andlumiglo chemiluminescent reagents (Kirkegaard and Perry, Mass.).

[0157] Kinase Assays.

[0158] Kinase immunoprecipitates were prepared as above in 1% TritonX-100 lysis buffer, given one wash in kinase buffer before incubation in50 μl of kinase buffer (20 mM HEPES pH 7.6, 10 mM MgCl₂) in the presenceof 4 μg of the synthetic substrate peptide raytide EL (Oncogene) andγ[³³p]-ATP for 15 min at room temperature. The kinase buffer containingthe labeled peptide was collected and loaded onto phosphocellulose paperdiscs. The paper was washed 3 times with 0.5% phosphoric acid to removeunincorporated ³³P-ATP and once with acetone, dried and counted in aβ-counter. Results are shown in arbitrary units and each represents oneof four independent experiments. The presence of Lck was determined byimmunoblotting lck immunoprecipitates run on non-reducing SDS page withanti-Lck (not shown).

[0159] Subcloning and Mutation of Zap-70, Cb1, EphB6 and EphB1

[0160] cDNAs for Zap-70, Cb1, EphB1, EphB6, ephrin-A1 and ephrin-B1 werecloned from normal human thymocyte RNA by RT-PCR into the expressionvector pcDNA3 (Invitrogen, CA) and sequenced. Mutants of these molecules(Zap:Y493F, Cb1 G306E, 70-Z) (EphB1: truncation of 102 C-terminal aminoacids, knase-null K651Q) (EphB6-DN: deletion of cytoplasmic tail) werecreated using the overlapping PCR technique to introduce the requiredbase changes, using cloned cDNAs as the template. Kinase-null B1 wascreated by mutating lysine 651 to glutamine (K651Q). The resulting cDNAswere cloned and sequenced to confirm the mutations. Myc-tagged versionsof EphB6 and of the truncated EphB1 receptor were generated by insertionof a Myc tag and constructs verified by sequencing. Expression of wildtype proteins and mutants were examined by transfection in COS-7 cellsand western blotting with appropriate antibodies. All mutations wereexpressed as well as respective wild types. The truncated form of EphB1was an active kinase, like the wild type protein. Kinase-null EphB1 hadno detectable kinase activity.

[0161] Transfection of Cell Lines.

[0162] Adherent COS-7, HEK 293 and NIH 3T3 cells were routinelytransiently transfected using the lipid reagent lipofectamine (LifeTechnologies, Grand Island, N.Y.). The DNA-lipid mixtures were appliedto the cells for 5 hours in the absence of serum, before the addition ofcomplete medium. Cells were given 72 hours to express the transfectedproteins before harvest.

[0163] To raise stable EphB6 overexpressing cells, the mature humanT-cell line jurkat was transfected with empty pcDNA3, EphB6-M, orDN-BphB6. The jurkat cells were electroporated in 400 μl complete RPMImedium with 30 μg of DNA by pulsing once for 65 msec at 260V (BTKelectro square porator, BTX, division of Genetronics Inc, San Diego,Calif.). Cells were incubated at 37° C. for 24 hours before addition ofG418 to the medium. After 30 days of selection the resulting oligoclonalcell populations were screened by immunoprecipitation with anti-MYC andwestern blotting with anti-myc or anti-EphB6 and the highest EphB6expressing cell population (B6-J) selected.

[0164] Isolation of Human Thymocytes.

[0165] Thymuses were obtained from children undergoing open heartsurgery. Mononuclear cells were isolated by Ficoll-hypaque gradientcentrifugation. Adherent cells were removed by incubation to plasticdishes for 60 minutes at 37° C. The resulting thymocytes aretypically >95% CD3.

[0166] Stimulation of EphB6 Receptor Transfected Cells with MembraneBound and Soluble Ligand.

[0167] To assay for stimulation with membrane bound forms of the ephrinligands, receptor-expressing cells were resuspended using 2.5 mM EDTAand after washing, overlaid on a confluent monolayer of control orligand-expressing cells. After incubation at 37° C. for 1 hour, all thecells were solubilized in 1% Triton lysis buffer. Soluble ephrin-B1-Fcfusion-protein dimer was purchased from R&D Systems (Minneapolis,Minn.). The dimeric ephrin-B1 fusion protein was pre-complexed withF(ab)′₂ goat anti-human Fc (pierce) to form oligomers. F(ab)′₂ goatanti-human Fc was used as a control (no stimulation) where necessary.Although murine ephrin-B1 was utilized, this effectively induced humanEphB6 phosphorylation.

[0168] Analysis of CD25 Expression by Flow Cytometry.

[0169] Cells were incubated in 0.5% serum for 24 hours with or without 5μg/ml soluble oligomerized or immobilized ephrin-B1 and immobilizedanti-CD3 antibody. Anti-human-CD19 antibody was used as an irrelevantprotein control for immobilized ephrin-B1 where necesssary. Theexpression of CD25 was then analyzed by staining with pre-labeledanti-CD25 and isotype control Immunotech).

Example 1

[0170] To determine if the catalytically inactive EphB6 receptor couldbe tyrosine phosphorylated in response to ephrin-B ligand stimulation,we transiently expressed human EphB6 in COS-7 cells and exposed thosecells to ephrin-B1. The EphB6 receptor was expressed as a C-terminalmyc-tagged protein (EphB6-M). To provide cell surface expressed ligands,we transfected COS-7 cells with pcDNA3 expression vector containingeither ephrin-A1 or ephrin-B1 cDNA. Ligand expression was verified byimmunoblotting (not shown). EphB6 receptor expressing cells wereoverlaid on cells transfected with ephrin-B1, ephrin-A1 or empty vector,and co-incubated for an hour at 37° C. The EphB6 receptor was thenprecipitated with anti-myc and immunoblotted with anti-phosphotyrosineantibody. Stimulation of EphB6 with ephrin-B1-expressing cells resultedin a major increase in EphB6 tyrosine phosphorylation, whileco-incubation with ephrin-A1-expressing or control cells had no effect(FIG. 1A). The increase in EphB6 receptor tyrosine phosphorylationcaused by co-incubation with ephrin-B1-expressing cells was alsoobserved upon transfection of NIH 3T3 fibroblasts and HEK 293 humanembryonic kidney cells (FIG. 1B), indicating the effect was not cellspecific. Stimulation of EphB6 receptor tyrosine phosphorylation wasboth time and ligand concentration dependent (FIGS. 1C,D).

[0171] In contrast to soluble monomers of ephrin, which can inhibit Ephreceptor signaling, dimerized or oligomerized forms can stimulatereceptor autophosphorylation and signaling (Davis et al. (1994); Sakanoet al. (1996)). A soluble dimerized form of the ephrin-B1 ligand wasalso found to induce EphB6 phosphorylation. Although recombinant murineephrin-B1 was utilized, it induced EphB6 phosphorylation as effectivelyas membrane expressed human ephrin-B1. Moreover, this ephrin-B1 inducedphosphorylation could be completely inhibited by the addition of solubleEphB6 receptor to the medium (FIG. 1E), strongly suggesting theexistence of a direct interaction between ephrin-B1 and EphB6 receptor.

Example 2

[0172] To demonstrate that EphB6 is trans-phosphorylated uponhetero-oligomerization with catalytically active members of the Ephfamily, EphB6 receptor was coexpressed with human EphB1 receptor inCOS-7 cells. The EphB1 receptor was found to be constitutively activatedwhen overexpressed. EphB6 underwent significant tyrosine phosphorylationupon coepression with the EphB1 receptor, trans-phosphorylated in amanner analogous to ErbB-3 (FIG. 2A). In contrast, catalyticallyinactive EphB1 (K651Q, B1-KD) was unable induce EphB6 phosphorylation(FIG. 2b). In NIH 3T3 fibroblasts, where the basal activity of EphB1 wasdetermined to be much lower than in 293 or COS-7 cells, EphB6trans-phosphorylation occurred in a ligand dependent manner (FIG. 2C).

[0173] As EphB1 and EphB6 have essentially the same electrophoreticmobility, the observed phosphorylation of EphB6 could, however, be dueto co-precipitating phosphorylated EphB1 in this over-expressing system.Therefore, to unambiguously distinguish between the two receptors, amyc-tagged truncated EphB1 receptor lacking 102 C-terminal residues wasconstructed, but with its kinase domain intact (B1-Tr). Like the wildtype receptor, truncated EphB1 was constitutively tyrosinephosphorylated, but now clearly smaller than EphB6. Co-expression oftruncated EphB1 also resulted in dramatically increased EphB6phosphorylation (FIGS. 2D,E), demonstrating that EphB6 phosphorylationcan be provided by a catalytically active EphB receptor and suggestingthat ephrin-B1 induced EphB6 phosphorylation may similarly result fromtrans-phosphorylation.

Example 3

[0174] In RT-PCR analysis, we detected EphB6 expression in humanthymocytes as well as in mature peripheral blood T cells and in the Tcell line Jurkat (FIG. 3). Two catalytically active members of the Ephfamily, EphB1 and EphB2, were also found to be expressed throughout theT cell lineage, while EphA2 could only be detected in thymocytes. Thepersistent expression of EphB6 across the T cell lineage suggested itmight be important both during differentiation and in mature T cellfunction.

[0175] Single cell suspensions of human thymocytes were stimulated withanti-CD3 for 10 minutes, the receptor immunoprecipitated with anti-EphB6antibodies and blotted with anti-phosphotyrosine. Polyclonal antibodiesto EphB6 were raised against a peptide from the extreme C-terminal ofEphB6, a unique sequence not present in any other known Eph receptor(see Experimental Procedures). This efficiently immunoprecipitated andWestern blotted myc-tagged EphB6 (not shown). While tyrosinephosphorylation of the EphB6 receptor itself was not detected inresponse to anti-CD3 stimulation, a tyrosine phosphorylated protein ofapproximately 115 kDa (pp115) was co-precipitated (FIG. 4A).

[0176] pp115 was the only highly tyrosine phosphorylated proteinconsistently associated with EphB6 and remained for at least 20 minutesafter anti-CD3 stimulation (FIG. 4B). The electrophoretic mobility ofpp115 appeared similar to that of the c-Cb1 proto-oncogene, which ishighly phosphorylated after TCR/CD3 stimulation Tsygankov et al. (1996))(FIG. 4C). Western blotting with anti-EphB6 revealed the presence of aband of the expected molecular weight in Cb1 immunoprecipitates, but notin immunoprecipitates of FAK or Vav (FIG. 4D). Preimmune serum controlblotting was also negative. This association was not noticeably alteredby addition of anti-CD3, indicating that TCR/CD3 stimulation primarilyinduced Cb1 phosphorylation, rather than increasing its recruitment tothe EphB6 receptor. Cb1 is central to signaling pathways from manyreceptors, functioning as a regulator of receptor tyrosine kinaseactivity, through initiation of receptor ubiquitination, and induciblybinding a variety of signal transducing molecules (Tsygankov et al.(1996); Fournel et al. (1996); Lupher et al. (1996); Lupher et al.(1997); Lupher et al. (1998); Ota et al. (1997); Thien et al. (1999);van Leeuwen (1999); Lee et al. (1999); Levkowitz et al. (1998); Miyakeet al. (1998)).

[0177] This was confirmed by co-expressing human Cb1 with either theEphB6 or EphB1 receptor in COS-7 cells. Cb1 appeared to specificallyco-precipitate with EphB6, as association with the catalytically activereceptor EphB1 coud not be detected (FIG. 4E). Stimulation withephrin-B1 expressing cells did not alter the level of EphB6-Cb1association, nor Cb1 tyrosine phosphorylation (not shown). To furthercharacterize this interaction, the binding of two mutants of Cb1 toEphB6 were examined. The first, G306E (Cb1*) was initially identified asa mutation in the C.elegans Cb1 orthologue, sli-1 (Jongeward et al.(1995)) and causes loss of Cb1 binding to the ErbB1 and PDGF receptorsby disruption of the Cb1 phosphotyrosine binding domain (Bonita et al.(1997); Thien et al. (1997)). The second, 70-Z Cb1 (Cb1**), isolated asan oncogene from a murine B cell line (Blake et al. (1991)), contains aninternal 17 amino acid deletion in the Cb1 RING finger domain While the70-Z mutation only slightly decreased Cb1 binding to the EphB6 receptor,the G306E point mutation completely abolished association (FIG. 4F);confirming the specificity of binding and drawing a parallel between Cb1binding to the EGP and PDGF receptors and its association with EphB6.

Example 4

[0178] Co-expression of Zap-70 with EphB6 or EphB1 receptors in COS-7cells revealed a selective downregulation of Zap-70 phosphorylation byEphB6 (FIG. 5A). The EphB6 receptor inhibited Zap-70 tyrosinephosphorylation, while no significant change was observed upon EphB1co-expression. This effect was ligand responsive, as a further decreasein Zap-70 phosphorylation occurred upon incubation of EphB6co-transfected cells with ephrin-B1 expressing cells (FIG. 5A). Theinduction of EphB6 receptor tyrosine phosphorylation by ephrin-B1probably contributes to the inhibition of Zap-70 through increasedrecruitment of effector proteins to the receptor.

[0179] Stimulation of the TCR complex leads to Zap-70 kinasephosphorylation by the Fyn and Lck src-like tyrosine kinases andsubsequent Zap-70 activation, primarily through phosphorylation oftyrosine residue 493 in the Zap-70 catalytic domain (Wange et al. (1995)Mege et al. (1996); Kong et al. (1996)). The removal of Y493 results ina level of Zap-70 phosphorylation essentially reflecting its own basalkinase activity. A Y493F Zap-70 mutant (Zap*) was constructed and whiledemonstrating significantly lower tyrosine phosphorylation than wildtype, Y493F Zap-70 was unaffected by EphB6 co-expression (FIG. 5B). Thissuggested that EphB6 might specifically affect phosphorylation oftyrosine residues characteristic of activated Zap-70.

[0180] The ability of EphB6 to alter signaling in T cells wasdemonstrated as follows. Stable overexpression of the myc-tagged EphB6receptor in the mature T cell line Jurkat (B6-J) (FIG. 5C) wasestablished. The transfected EphB6 receptor appeared to be functional,undergoing tyrosine phosphorylation upon stimulation of transfectedT-cells with ephrin-B1 (FIG. 5D). TCR surface expression on control andB6-J cells was found to be equivalent by staining with anti-CD3ε-FITC.

[0181] Zap-70 was immunoprecipitated from control and EphB6 transfectedT cells and its phosphorylation status examined by Western blotting. Inagreement with our previous results, expressing Y493F Zap-70 in 293cells, the basal phosphorylation of Zap-70 was not significantlyaffected by EphB6 overexpression. However, the induction of Zap-70phosphorylation in response to TCR/CD3 stimulation was stronglyinhibited (FIG. 5E). The amount of phosphorylated CD3ζ chain associatedwith Zap-70 was also decreased by EphB6 overexpression (FIG. 5F). Thesrc-family kinase Lck is primarily responsible for phosphorylation ofthe CD3ζ chain upon TCR stimulation and subsequently regulates Zap-70recruitment to the CD3 receptor complex, in addition to its activationof Zap-70 by direct phosphorylation. Lck activation by the CD45phosphatase is one of, if not the, earliest events following TCRligation and Lck kinase activity was significantly elevated after 5minutes anti-CD3 stimulation of the control Jurkat cells. However, Lckactivation was constitutively inhibited in EphB6 overexpressing B6-Jcells (FIGS. 6A,B). Ephrin-B1 treatment of anti-CD3 stimulated controlcells also partially inhibited Lck activation, but had no further effectupon Lck in EphB6 overexpressing cells, either alone, or in thenpresence of anti-CD3. The absence of ligand effect in B6-J cellssuggests that the basal activity of overexpressed EphB6 alone issufficient to prevent Lck activation. In sum, these results indicatethat the EphB6 receptor modulates TCR signaling, by regulating thetyrosine phosphorylation and activity of TCR-associated kinases.

Example 5

[0182] The EphB6 receptor could downregulate both Lck and Zap-70kinases, suggesting that EphB6 inhibition of TCR mediated CD25upregulation was demonstrated as follows. pcDNA3 control and B6-J Jurkatcells were stimulated with anti-CD3 and the EphB6 ligand ephrin-B1. Theligand had no effect upon resting control cells, and demonstrated only asmall and variable inhibition of TCR mediated CD25 upregulation (seeFIG. 7A). In contrast, overexpression of EphB6, although variablyaffecting the basal level of CD25 expression, completely inhibited theability of TCR stimulation to induce CD25 upregulation (FIG. 7B). Inparallel with the ability of EphB6 to inhibit lck activation, no furtherinhibition of CD25 expression was observed upon addition of theephrin-B1 ligand.

[0183] EphB6 is naturally highly expressed in thymocytes and wheninduction of CD25 in response to TCR activation was examined in thesecells, ephrin-B1 co-stimulation caused a strong inhibition of CD25upregulation, while ephrin-B1 alone had little effect (see FIG. 8C).

[0184] To confirm this role of endogenous EphB6, Jurkat cell linesoverexpressing a dominant-negative form of EphB6, namely eliminating thecytoplasmic domain of the receptor (DN-J) were created. In constrast tooverpression of wild type EphB6, the dominant-negative receptor did notprevent anti-CD3 mediated induction of CD25 expression and a furtherenhancement of anti-CD3 induced upregulation of CD25 was observed uponephrin-B1 co-stimulation; presumably due to the removal of inhibitoryinput from the endogenous EphB6 receptor (see FIGS. 8A and 8B).

[0185] In summary, the results from Examples 1-5 provide support formethods of modulating T cells by suppressing antigen induced CD25expression through manipulation of EphB6 receptors. The followingExamples 6-8 further support methods of modulating the immune systemthrough manipulation of EphB6 by demonstrating that modulation of EphB6provides a method to modulate antigen induced cell death (AICD).

Discussion of Examples 1-5

[0186] Although the structure of EphB6 is typical of the EphB receptors,its kinase domain contains numerous alterations to critical catalyticresidues and neither murine nor human EphB6 demonstrates kinase activity(Gurniak et al. (1996); Matsuoka et al. (1997)). Despite thesestructural abnormalities the human EphB6 receptor responds to ephrin-B1stimulation by undergoing tyrosine phosphorylation. Further, the EphB6tyrosine phosphorylation can be provided by a catalytically activepartner, in particular, by the EphB1 receptor. While EphB1 receptor cantrans-phosphorylate EphB6 upon co-transfection, in vivo EphB6 maypotentially interact with multiple members of the EphB sub-family.Lacking catalytic activity, EphB6 is unlikely to operate as anindependent receptor, but rather as part of a hetero-oligomeric complexwith the active EphB receptors. Catalytically active EphB1 and EphB2 areboth co-expressed with EphB6 throughout the T cell lineage, raising thepossibility that EphB6 may interact with both receptors.

[0187] Until now, ErbB-3 of the EGF receptor family (Pinkas et al.(1996)) has been the only example of a trans-phosphorylatedkinase-inactive receptor. However, without wanting to be bound by anyparticular theory, our findings suggest that this is a universalmechanism for signaling through catalytically inactive receptor tyrosinekinases. Two other kinase-inactive orphan receptors, Klg and Vik, (Chouet al. (1991); Hovens et al. (1992); Kelman et al. (1993); (Paul et al.(1992); Tamagnone et al. (1993); Stacker et al. (1993)) may signal in asimilar manner and have catalytically active partners, as yetundescribed. ErbB-3 acts to modulate the intensity and duration ofsignaling by its active partner (Pinkas et al. (1996); Levkowitz et al.(1998)) and trans-phosphorylation results in recruitment of Shc andphosphatidylinositol 3-kinase specifically to the ErbB-3 receptor chain(Kim et al. (1994); Waterman et al. (1999)). In similar fashion, thecatalytically inactive EphB6 receptor may recruit specific cytoplasmicsignaling molecules, as Cb1 appears to specifically bind EphB6 and notan active EphB1 partner (FIG. 4E).

[0188] Unusually for a receptor tyrosine kinase, and particularly for anEph receptor, EphB6 is most highly expressed in the thymus (Gurniak etal. (1996)). Several lines of evidence suggested a potential role forEphB6 in modulation of T-cell responses. First, several Eph familymembers interact with the src-like kinase Fyn (Choi et al. (1999); Elliset al. (1996); Hock et al. (1998)), a TCR-associated kinase critical forthe development of T-cell responses (Utting et al. (1998)). Secondly,Eph receptors can regulate re-organization of the actin cytoskeleton(Meima et al. (1997); Meima et al. (1997a)), an important event in TCRsignaling; as disruption of actin with cytochalasin D or Clostridiumbotulinum toxin inhibits T lymphocyte responses to antigen (Valitutti etal. (1995). And finally, the Eph receptors can modulateintegrin-mediated cell attachment (Becker et al. (2000); Huynh-Do et al.(1999)), integrins functioning as TCR co-receptors to modulate responsesin both mature T cells and thymocytes (Abraham et al. (1999); Bleijs etal. (1999); Ticchioni et al. (1993); Wulfing and Davis (1998);Vivinus-Nebot et al. (1999)).

[0189] In accordance with this hypothesis, it was shown that both Zap-70and Lck stimulation were decreased upon overexpression of EphB6 in Tcells (FIGS. 5 and 6). EphB6 did not affect a Zap-70 mutant lacking theactivating tyrosine 493 residue when co-pressed in COS-7 cells,appearing to prevent phosphorylation primarily of residuesphosphorylated in the activated state. Therefore, decreased Zap-70phosphorylation in COS-7 cells most likely reflects inhibition ofendogenous src-family kinases; while in Jurkat, the primary inhibitionof Lck activation by EphB6 is probably responsible for the absence ofZap-70 stimulation. This inhibition would reflect both a lack ofphosphorylated CD3ζ chain to recruit Zap-70 to the signaling complex,and a decrease in the direct modification of Zap-70 by Lck. Withoutwishing to be bound by any particular theory, the decrease in TCRstimulated Lck kinase activity is in all probability the consequence ofEphB6 induced re-arrangement of the cytoskeleton, sequestering lck awayfrom the TCR/CD3 receptor complex. In support of our hypothesis,stimulation of β1-integrins with either soluble ligand or antibody haspreviously been shown to inhibit TCR mediated activation of Lck andZap-70 (Mary et al. (1999)).

[0190] Inhibition of the early events of TCR signaling by overexpressionof EphB6 was found to ultimately translate into an inhibition of T cellresponse, such as the induction of CD25 (IL-2Rα) expression Expressionof CD25 is essential in the IL-2 driven clonal expansion that occursupon exposure to antigen. Failure to express the high affinity IL-2Rcomplex composed of the α, β and γ chains prevents the development ofthe necessary IL-2 autocrine proliferative loop. Thus without wishing tobe bound by any one theory, one of the biological functions of EphB6, inconjunction with other EphB receptors, may be to control the clonalexpansion of antigen activated T cells by suppressing antigen inducedCD25 expression and associated events. Several alternative models ofEphB6 function also become apparent. Under more physiologicalconditions, ligation of the EphB6 receptor may serve to maintainactivation of the TCR signaling pathway below a certain threshold,preventing premature activation by inappropriate low affinity TCRinteractions. Or, alternatively, the presence of varying ephrin-Bligands may modify the ability of T cells to respond to antigenspresented on different cell-surfaces.

[0191] EphB6, like the ErbB1 and PDGF receptors, was found to physicallyassociate with Cb1. The G315E mutation of the C.elegans Cb1 orthologueSli-1 prevents interaction with the nematode ErbB protein (let-23)(Jongeward et al. (1995)) and the analogous Cb1 mutation disruptsbinding to PDGF and ErbB-1 receptors (Bonita et al. (1997); Thien et al.(1997)). This mutation also abolished Cb1 association with EphB6 (FIG.4F). The G306E mutation disrupts the Cb1 phosphotyrosine-binding domain,suggesting that phosphorylation of EphB6 or an intermediate dockingprotein may be important for Cb1 binding to the receptor. Although EGFstimulation of the ErbB-1 receptor induces tyrosine phosphorylation ofCb1 (Levkowitz et al. (1996)), increased Cb1 phosphorylation uponstimulation of the EphB6 receptor with ephrin-B1 (not shown) was notdetected. This lack of Cb1 phosphorylation probably reflects the absenceof EphB6 catalytic activity, suggesting that EphB6 may simply recruitCb1 to the cell membrane, rather than modifying its function byphosphorylation. The failure to observe Cb1 phosphorylation uponephrin-B1 stimulation also suggests that it is not a substrate of thecatalytically active EphB6 partner. TCR/CD3 stimulation of T cellsresulted in phosphorylation of EphB6-associated Cb1, although EphB6itself did not undergo detectable phosphorylation, suggesting that Cb1phosphorylation is probably mediated by TCR/CD3 associated cytoplasmickinases.

[0192] The ability of Cb1 to bind EphB6 raises the possibility thatEphB6 expression may be regulated by Cb1 mediated modification. It isnow clear that Cb1 is responsible for the physical downregulation ofmany receptors through induction of receptor ubiquitination (Lee et al.(1999); Levkowitz et al. (1998); Miyake et al. (1998)). The addition ofmultiple ubiquitin moieties to the lysine residues of a protein targetsit for degradation, either in cytoplasmic proteasomes or the lysosomalcompartment (Hershko et al. (1998)). As all receptors binding to Cb1undergo ubiquitination, it is likely that EphB6 will be similarlyregulated. This activity of Cb1 is normally accompanied by itsphosphorylation, suggesting that anti-CD3 induced phosphorylation ofEphB6-associated Cb1 may trigger EphB6 downregulation (FIG. 8). WhileEphB6 may maintain activation of the TCR signaling pathway below acertain threshold, preventing premature responses to inappropriatestimulus, elimination of the inhibitory EphB6-Cb1 complex from theplasma membrane may be an obligatory event for maximal activation of theTCR signaling pathway.

[0193] To this point, Eph receptor function has been addressed primarilyin the development and function of the nervous system, where they wereshown to participate in targeting neurons and growth cones, as well asin synapse formation (Zhou et al. (1998); Flanagan et al. (1998)). Thisbiological activity is essentially due to the ability of Eph receptorsto reorganize the actin cytoskeleton and to control cell attachment byregulation of integrin receptors (Becker et al. (2000); Holland et al.(1997); Huynh-Do et al. (1999). Proper activation of T lymphocytes byantigen-presenting cells requires stimulation not only of the TCR, butthe combined and coordinated engagement of its co-receptors. Most TCRco-receptors bind cell-surface ligands and are concentrated in areas ofcell-cell contact, forming what has been termed an immunological synapse(Grakoui et al. (1999); Dustin et al. (1999)). Assembly of thesesynapses and subsequent T cell responses are strictly dependent uponcell attachment 101, actin cytoskeleton re-organization (Holsinger etal. (1998); Valitutti et al. (1995); Wulfing and Davis (1998)) andintegrin receptor signaling (Abraham et al. (1999); Bleijs et al.(1999); Ticchioni et al. (1993); Wulfing et al. (1998); Vivinus-Nebot etal. (1999)). Without wishing to be bound by any particular theory, EphBreceptors and in particular EphB6, may be involved in coordination of Tcell attachment and formation of the immunological synapse and thus maybe important modulators of both thymocyte selection and T-cellresponses.

Example 6

[0194] To explore the potential role of EphB6 in regulation of AICD, wegenerated stable expression of myc-tagged human EphB6 in the mature Tcell line Jurkat (FIG. 9A); a commonly used model of pre-stimulatedmature T cells in AICD studies. AICD was induced in EphB6 and controlpcDNA3 transfected cells by overnight stimulation with immobilizedanti-CD3 antibody. To activate the EphB6 receptor, cells were alsotreated with the EphB6 ligand, ephrin-B1. Stable overexpression of theEphB6 receptor was found to significantly enhance TCR-mediated apoptosisin a ephrin-B1-dependent manner (FIG. 9B), thus confirming its potentialto regulate the induction of AICD.

Example 7

[0195] Active T cell apoptosis is driven by the antigen-inducedexpression of the FASL and TNF death cytokines. In resting cells, bothFAS-L and TNF are weakly induced by TCR stimulation, but inpre-activated cells these cytokines are highly expressed uponstimulation. In vitro experiments suggest that CD4⁺ cells are primarilyeliminated by FAS-L, while AICD of CD8 cells is predominantly triggeredby TNF. We therefore examined the production of TNF by the control andEphB6 overexpressing CD4⁺ jurkat cells. Ephrin-B1 alone did not induceTNF product nor did it significantly alter the response to anti-CD3.Anti-CD3 stimulation induced significant TNF production in EphB6overexpressing Jurkat but not in control cells (FIG. 10). Thus, theincreased apoptosis observed in EphB6 overexpressing cells may be due inpart to increased TNF production.

Example 8

[0196] TNF efficiently activates both TNFR-I and TNFR-II. However,previous studies have suggested that only TNFR-I is coupled to a caspasecascade (Kozlosky et al. (1995)) and thus it may be the predominanttransmitter of the TNF apoptic signal (Daniel et al. (1996); O'Leary andWilkinson (1999)). We therefore examined the expression of the two TNFreceptors an EphB6 and control Jurkat cells. Expression of TNFR-II, butnot of TNFR-I, was suppressed upon incubation of both control and EphB6overexpressing cells with ephrin-B1 (FIG. 11). Activation of the TCRoverrode this effect in control cells, maintaining high TNFR-IIexpression despite ephrin-B1 stimulation. However, overexpression ofEphB6 maintained the ephrin-B1-induced down regulation of TNFR-II in thepresence of anti-CD3 stimulation. Interestingly, anti-CD3 stimulation ofEphB6 overexpressing cells also reduced TNFR-II expression, while it hadno effect upon the receptor in control cells. This is probablyresponsible for the greater degree of anti-CD3 induced apoptosisobserved in EphB6 overexpressing cells and suggests that the basalactivity of the EphB6 receptor is sufficient to make the cells moresensitive to the induction of apoptosis. Activation of the EphB6receptor by ephrin-B1 co-stimulation with anti-CD3 resulted in a furtherdecrease in TNFR-II expression, which is reflected in an increase ininduction of apoptosis.

[0197] TNFR-I and TNFR-II employ only partially distinct signalingpathways, both initiating the n-terminal JUN kinase cascade (Kozlosky etal. (1995)). Activation of the JNK pathway is required to protect cellsfrom TNF-mediated apoptosis (Adams et al. (1999); Wang et al. (1998)).We examined the influence of EphB6 upon the JNK cascade by following thethreonine-tyrosine phosphorylation (Thr183/Tyr185) of JNK upon anti-CD3and ephrin-B1 stimulation. Overexpression of EphB6 not only resulted inan alteration in the balance of TNFR expression in favor of TNFR-I, butit also strongly inhibited long-term anti-CD3 induced JNK stimulation(FIG. 12). This effect was highly specific, as none of the otherpotentially anti-apoptic pathways examined, including AKT activation andBc1-2 expression, was affected. the suppression of JNK activationappeared to be ligand-independent, suggesting that the basal activity ofoverexpressed EphB6 was sufficient for JUN kinase inhibition. theelimination of JNK-JUN signaling was previously reported to enhanceTNF-induced apoptosis (Adams et al. (1999); Wang et al. (1998)),suggesting that the selective inhibition of the JUN kinase pathwayobserved here could further the promotion of AICD by EphB6.

[0198] Interestingly, while addressing the role of EphB6 in apoptosis,we observed that overexpression of a dominant negative form of EphB6(cytoplasmic domain deleted) also increased the induction of AICD. Thisis surprising in light of the ability of wild type EphB6 to also promoteAICD and probably reflects the ability of the DN receptor to enhance TCRmediated responses, as observed when examining CD25 expression. Whilenot wishing to be bound by any particular theory, this presumably as theresult of removing TCR inhibitory input from the endogenous EphB6receptor. However, this effectively overrides the actual inhibition ofEphB6-specific apoptic effects, such as TNFR modulation and TNFproduction, by the dominant negative receptor. While the apopticcontribution of the endogenous EphB6 receptor is removed, apoptosisstill appears to increase due increased sensitivity to induction throughthe TCR.

[0199] In sum, our findings conclusively demonstrate that the EphB6receptor serves an important role as a TCR co-receptor in the inductionof AICD in mature activated T cells. Although driven by differentfactors to AICD, and proceeding via a different mechanism, the negativeselection of thymocytes is also predominantly a TCR induced apopticprocess. As the EphB6 receptor is strongly expressed in thymocytes, itis therefore likely that it also has an important role in regulatingnegative selection.

Materials and Methods for Examples 6-8 Western Blotting

[0200] Cells were quickly resuspended in ice cold lysis bufferconsisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%Triton X-100, 1 mM ethylene glycol-bis(β-aminoethylether)-N,N,N′-N′-tetraacetic acid (EGTA), 10 μg/mlleupeptin, 10 μg/ml aprotinin, 1 mM PMSF, 1 mM Na-orthovanadate and 50mM NaF. After solubilization an ice for 15 minutes, debris was removedby centrifugation at 12,000 g for 10 minutes at 4° C. and SDS samplebuffer added. Samples were separated on SDS-polyacrylamide gels andtransferred to nitrocellulose membranes (Amersham, Arlington Heights,Ill.). Membranes were blocked overnight at 4° C. with 7% blotting gradenon-fat milk (Biorad, Richmond, Calif.) in PBS. Immunoblottingantibodies were added at optimal dilutions in PBS-T or TBS-T (0.1%Tween-20) and incubated at 4° C. overnight. After extensive washing withPBS-T, bound antibodies were detected using horseradish-peroxidaseconjugated donkey anti-rabbit or sheep anti-mouse antibodies (Amersham,Arlington Heights, Ill.) and LumiGlo chemiluminescent reagents(Kirkegaard and Perry, Mass.).

[0201] Stoning of EphB6

[0202] cDNA for EphB6 was cloned from normal human thymocyte RNA byRT-PCR into the expression vector pcDNA3 (Invitrogen, Calif.) andsequenced. Myc-tagged version of EphB6 was generated by insertion of anin frame Myc tag and construct verified by sequencing. Expression of thetagged protein was examined by transfection in COS-7 cells and Westernblotting with appropriate antibodies.

[0203] EphB6 Stable Expression

[0204] To raise stable EphB6 expressing cells, the mature human T-cellline Jurkat was transfected with empty pcDNA3 or EphB6-M. The jurkatcells were electroporated in 400 μl complete RPMI medium with 30 μg ofDNA by pulsing once for 65 msec at 260 V (BTK electro square porator,BTX Division of Genetronics Inc, San Diego, Calif.). Cells wereincubated at 37° C. for 24 hours before addition of G418 to the medium.After 30 days of selection expression of the EphB6 receptor in resultingcell population was confirmed by immunoprecipitation with anti-Myc andwestern blotting with anti-Myc or anti-EphB6.

[0205] Stimulation of EphB6 Overexpressing and Control Cells

[0206] Soluble ephrin-B1-Fc fusion-protein dimers were purchased fromR&D Systems (Minneapolis, Minn.). The dimeric ephrin-B1 fusion proteinwas pre-complexed with F(ab)′₂ goat anti-human Pc (Pierce) to formoligomers. F(ab)′₂ goat anti-human Fc was used as a control (nostimulation) where necessary. Although murine ephrin-B1 was used thiseffectively induced human EphB6 phosphorylation. Anti-CD3 (PharMingene,Canda) were immobilized on 24-well plates at 20 μg/ml for 4 hours atroom temperatue, plates were rinsed are with PBS and cells stimulatedfor 24 hors, 37° C.

[0207] Analysis of TNFR I and TNFR II Expression by Flow Cytometry

[0208] EphB6 and pcDNA3 Jurkat cells were incubated in 0.5% seran for 24hours with or without 5 μg/ml soluble oligomerized ephrin-B1 andimmobilized anti-CD3 antibody. The expression of TNFα, TNFR I and TNFRII were then analyzed by staining with corresponding PE-labeled antibodyand isotype control. Anti-TNFR-I and anti-TNFR II were from R&D Systems,MN.

[0209] Analysis of Apoptosis

[0210] Cells were resuspended in RPMI medium with 0.5% serum andsupplements as indicated. After 24 hours incubation the percentage ofapoptic cells was assessed by Annexin-V-FITC (Boehringer Mannheim,Indianapolis, Ind.) binding and Propidium iodide (PI) staining. Cellswere analyzed on an Epics Elite V Flow Cytometer (Coulter Electronics).

[0211] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0212] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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[0339] Zhou, R. The Eph family receptors and ligands. [Review] [181refs]. Pharmacology & Therapeutics 77, 151-81 (1998).

[0340] Zou, J. X. et al. An Eph receptor regulates integrin activitythrough R-Ras. Proc Natl Acad Sci USA 96, 13813-8 (1999).

DETAILED FIGURE LEGENDS

[0341]FIG. 1. Tyrosine Phosphorylation of the EphB6 Receptor is Inducedby Ephrin-B1 Ligand Stimulation.

[0342] (a) COS-7 cells transiently transfected with EphB6-M encodingexpression vector (pcDNA3) were stimulated by co-incubation for 1 hourat 37° C. with COS-7 cells transfected with empty vector (−), ephrin-A1(A1), or ephrin-B1 (B1) cDNAs in pcDNA3. Receptor phosphorylation wasmonitored by immunoblotting anti-Myc immunoprecipitates withanti-phosphotyrosine (PY). EphB6-M expression was determined by blottingwith anti-Myc.

[0343] (b) HEK-293 and NIH 3T3 cells transiently expressing EphB6-M wereco-incubated for 1 hour with ligand expressing HEK-293 or NIH 3T3 cellsrespectively and receptor tyrosine phosphorylation and expression levelsdetermined as above.

[0344] (c) Time dependent phosphorylation of EphB6. EphB6-M-expressingCOS-7 cells were co-incubated with ephrin-B1 transfected COS-7 cells forthe indicated time periods. EphB6-M receptor phosphorylation andexpression were determined as in (a).

[0345] (d) Ligand concentration dependent EphB6 phosphorylation.EphB6-M-expressing COS-7 cells were co-incubated for 1 hour with COS-7cells transfected with 5 μg of pcDNA3 (−), or varying amounts ofephrin-B1-pcDNA3 (B1) as indicated.

[0346] (e) Soluble EphB6 receptor blocks ephrin-B1 induced EphB6phosphorylation. Control (pcDNA3) or EphB6-M transfected cells werestimulated with 1 μg/ml soluble oligomerized ephrin-B1 (B1) in thepresence (+B6-R) or absence of 5 μg/ml soluble EphB6 receptor for 30minutes at 37° C. Cells were lysed by boiling in 1% SDS. Phosphorylationof the membrane expressed myc-tagged EphB6 receptor was examined byimmunoprecipitation with anti-phosphotyrosine and Western blotting withanti-Myc.

[0347]FIG. 2. EphB6 Receptor Phosphorylation is Induced by Co-expressionof Catalytically Active EphB1 Receptor.

[0348] (a) COS-7 cells transiently transfected with either EphB1,EphB6-M (B6-M), or both receptors were co-incubated for 1 hour at 37° C.with control (−) or ephrin-B1 expressing (+) cells. Cells were lysed andimmunoprecipitation performed with anti-phosphotyrosine. The presence ofphosphorylated EphB6-M was detected by immunoblotting with anti-Myc.EphB1 and EphB6-M expression levels were quantitated by Western blottingwith anti-EphB1 and anti-Myc, respectively.

[0349] (b) COS-7 cells were transiently transfected with EphB6-M (B6-M),EphB1, T-7 tagged kinase inactive EphB1 (B1-KD) or co-transfected withEphB6-M and EphB1 or B1-KD. After 72 hours the cells were lysed andimmunoprecipitation performed with anti-phosphotyrosine. The presence ofEphB6-M in immunoprecipitates was detected as in (a). Expression of thetransfected proteins was examined by western blotting.

[0350] (c) NIH 3T3 cells were transiently transfected with EphB6-Malone, or in combination with EphB1. Cells were stimulated with 1 μg/mlof soluble oligomerized ephrin-B1, lysed and EphB6 receptor precipitatedwith anti-Myc. Phosphorylation of EphB6-M was monitored byimmunoblotting with anti-phosphotyrosine.

[0351] (d) Truncated EphB1 receptor induces phosphorylation of EphB6.COS-7 cells transiently transfected with EphB6-M, truncated myc-taggedEphB1 (B1-Tr), or both receptors, were incubated for 1 hour with control(−) or ephrin-B1 ligand expressing (+) COS-7. Cells were lysed andprecipitation with anti-phosphotyrosine performed. The presence ofEphB6-M in immunoprecipitates and the EphB6-M expression level weredetermined by anti-Myc Western blot, as in (a).

[0352] (e) Eph6-M and B1-Tr were expressed in COS-7 cells as indicatedand analyzed as in (c).

[0353]FIG. 3. Eph Receptor Expression in Human Thymocytes and T cells.

[0354] Expression of the EphA1, EphB1, EphB2 and EphB6 receptors wasexamined by RT-PCR in human thymocytes, peripheral blood T-lymphocytesand the mature T-cell line Jurkat. Control βactin primers were includedin each reaction. The expected product sizes are: βactin—660 bp,EphA1—279 bp, EphB1—309 bp, EphB2—375 bp, EphB6—294 bp. The identity ofthe PCR products was confirmed by sequencing. Water controls (no DNA)were all negative (not shown). A 100 bp size ladder is shown on theright (Gibco, BRL).

[0355]FIG. 4. EphB6 Associates with c-Cb1.

[0356] (a) pp115 co-precipitates with EphB6 in human thymocytes:Thymocytes were stimulated with 1 μg/ml anti-CD3 in the presence of 5μg/ml crosslinking antibody for 10 minutes. Cells were then lysed andprecipitated with anti-EphB6 (358) or pre-immune serum (PI).Phosphorylated proteins in these complexes were detected by blottingwith anti-phosphotyrosine. Preimmune (PI) antisera did not precipitatephosphorylated pp115.

[0357] (b) Time course of pp115 association with EphB6. Thymocytes werestimulated with anti-CD3 for the indicated time periods, precipitatedwith anti-EphB6 (358) and blotted with anti-phosphotyrosine.

[0358] (c) pp115 has the same electrophoretic mobility as c-Cb1.Thymocytes were stimulated with 1 μg/ml anti-CD3 in the presence of 5μg/ml of crosslinking antibody for 10 min and Cb1 and EphB6immunoprecipitated. Immunocomplexes were resolved by SDS PAGE,transferred to the nitrocellulose and blotted with anti-phosphotyrosine.

[0359] (d) Cb1, Vav, and FAK were immunoprecipitated from thymocytelysates after anti-CD3 stimulation and immunocomplexes Western blottedwith anti-EphB6 (358) or pre-immune serum as indicated.

[0360] (e) EphB6, but not EphB1, co-precipitates with Cb1. COS-7 cellswere transiently transfected with Cb1 and EphB6-M (B6-M), or Cb1 andEphB1 as indicated. After 72 hours, Cb1 was precipitated and associationwith EphB6-M and EphB1 examined by blotting with anti-Myc and anti-EphB1respectively.

[0361] (f) The G306E loss-of-function Cb1 mutant does not bind EphB6.COS-7 cells were transiently transfected with wild type Cb1, G306E Cb1(Cb1*), or the oncogenic 70-Z Cb1 mutant (Cb1**), either alone or incombination with EphB6-M as indicated. After 72 hours EphB6-Massociation with Cb1 was examined by immunoblotting Cb1immunoprecipitates with anti-Myc. Expression of each form of Cb1 andEphB6 was confirmed by Western blotting of cell lysates.

[0362]FIG. 5. EphB6 Downregulates the Zap-70 Kinase.

[0363] (a) EphB6 downregulates Zap-70 tyrosine phosphorylation. Zap-70was transiently expressed in COS-7 cells, alone, or in combination withEphB6-M (B6-M) or EphB1 receptors. To activate EphB6, cells wereincubated for 1 hour with ephrin-B1 ligand (+) expressing cells. Zap-70phosphorylation was then analyzed by immunoblotting Zap-70immunoprecipitates with anti-phosphotyrosine.

[0364] (b) Phosphorylation of Y493F Zap-70 is not altered by EphB6.Zap-70 or Y493F Zap-70 (Zap*) were expressed in COS-7 cells, alone, orwith EphB6-M (B6-M. The phosphorylation status of Zap-70 and Zap* wereanalyzed by anti-phosphotyrosine blotting and expression by anti-Zap-70blot. EphB6 expression was determined by Western blot of lysates.

[0365] (c,d) Transfected EphB6-M is tyrosine phosphorylated in Jurkatupon stimulation with ephrin-B1. The mature human T-cell line Jurkat wastransfected with empty pcDNA3 or EphB6-M. After 30 days of Geneticinselection the resulting oligoclonal cell populations were screened byimmunoprecipitation with anti-Myc and western blotting with anti-Myc oranti-EphB6 and the highest expressing cell population (B6J) selected.B6-J and pcDNA3 Jurkat cells were stimulated with 1 μg/ml solubleephrin-B1 for 15 minutes at 37oC, cells lysed, EphB6-Mimmunoprecipitated with anti-Myc and its phosphorylation examined.

[0366] (e,f) Overexpression of EphB6 downregulates phosphorylation ofZap-70 and Zap-70 asssociated CD3ζ in Jurkat. Transfected Jurkat cellswere stimulated 1 μg/ml soluble dimerized ephrin-B1 for 15 minutes at37° C. and then costimulated for 7 minutes with 4 μg/ml anti-CD3. Zap-70and CD3ζ tyrosine phosphorylation was then examined byanti-phosphotyrosine Western blotting of kinase immunoprecipitates.Results shown represent four independent experiments.

[0367]FIG. 6. The EphB6 Receptor Inhibits TCR Induced Activation of Lck.

[0368] (a,b) Lck immunoprecipitates were prepared from pcDNA3 and B6-JJurkat cells stimulated as in FIG. 5e. Immunocomplexes were incubated in50 μl of kinase buffer in the presence of 4 μg of the syntheticsubstrate peptide Raytide EL and γ[32P]-ATP for 15 min at roomtemperature. The kinase buffer containing the peptide was collected andloaded onto phosphocellulose paper. The paper was washed 3 times with0.5% phosphoric acid and once with acetone, dried and counted in aβ-counter. Results are shown in arbitrary units and represent one offour independent experiments. The presence of Lck was determined byimmunoblotting of Lck immunoprecipitates run on non-reducing SDS PAGEwith anti-Lck (not shown).

[0369]FIG. 7. EphB6 Overexpression Prevents TCR Mediated Upregulation ofCD25.

[0370] (a,b) B6-J and pcDNA3 Jurkat cells were incubated in 0.5% serumfor 24 hours with or without 5 μ/ml soluble oligomerized ephrin-B1 andimmobilized anti-CD3 antibody as indicated. The expression of CD25 wasthen analyzed by staining with PE-labeled anti-CD25. The percentage ofCD25 expressing cells is given in each case after subtraction of theisotype control.

[0371]FIG. 8. Endogenous EphB6 Downregulates CD25 Upregulation.

[0372] (A). Dominant negative (DN) EphB6 receptor expressing Jurkatcells (DN-J) were generated as in FIG. 4A. Expression of the DN receptorwas assessed by Western Blot (see insert). DN-J cells were stimulated asin FIG. 7 and CD25 expression analyzed by flow cytometry. Resultsrepresent one of three independent experiments.

[0373] (B) A further view of dominant negative (DN) EphB6 receptorexpressing Jurkat cells (DN-J) were generated as in FIG. 4A. Expressionof the DN receptor was assessed by Western Blot (see insert). DN-J cellswere stimulated as in FIG. 7 and CD25 expression analyzed by flowcytometry. Results represent one of three independent experiments.

[0374] (C). Purified thymocytes were starved for 24 hours, resuspendedin 0.5% serum and stimulated with plate-immobilized anti-CD3 andephrin-B1 as indicated. Expression of CD25 was analyzed by flowcytometry upon staining with PE-labeled anti-CD25 antibody. Thepercentage of CD25 expressing cells is given after subtraction of theisotype control. Results represent one of three independent experiments.

[0375]FIG. 9. The EphB6 receptor enhances TCR mediated apoptosis. a,Stable expression of EphB6 receptor. The mature T cell line Jurkat wastransfected with empty peDNA3 expression vector or myc-tagged EphB6.After 30 days of Geneticin selection EphB6 expression in the selectedcells was confirmed by immunoprecipitation with anti-myc and blottingwith either anti-myc or anti-EphB6. Equivalent expression of TCR/CD3 onEphB6 and control cells was confirmed by flow cytometry (not shown). b,EphB6 overexpressing (B6-13) and control pcDNA3 transfected cells wereincubated in 0.5% serum for 24 hours with or without 5 μg/ml solubleoligomerized ephrin-B1 (B1) and immobilized anti-CD3 antibody asindicated. Induction of apoptosis was analyzed by annexin-V binding. Thepercentage of apoptic cells is given in each case. The results shownrepresent four independent experiments.

[0376]FIG. 10. The EphB6-dependent increase in activation induced celldeath is accompanied by increased TNFα production EphB6 overexpressingand control Jurkat cells were stimulated for 24 hours as in FIG. 1. TNFαproduction was quantitated by chemiluminescent immunoassay of the cellculture supernatant.

[0377]FIG. 11. The EphB6 receptor inhibits expression of TNFR II but notTNFR I. Control (a) and EphB6 overexpressing (b) Jurkat T cells werestimulated as in FIG. 1 and expression of TNFR I and TNFR II determinedby staining with PE-labeled anti-TNFR I or anti-TNFR II antibodiesaccordingly. TNFR I and TNFR II expression is given in arbitrary units(AU) after subtraction of the isotype control. The results shownrepresent three independent experiments.

[0378]FIG. 12. The EphB6 receptor prevents activation of p54 JNK. EphB6and pcDNA3 control cells were stimulated as in FIG. 1. Cells were lysed,clarified by centrifugation and the lysates resolved by SDS PAGE.Phosphorylation of Jun kinase JNK) and Akt and expression of Bcl-2, wereanalyzed by Western Blotting with the appropriate antibody as indicated.The results shown represent three independent experiments.

[0379]FIG. 13. Model of EphB6 Receptor Interaction with the TCRSignaling Pathway. Binding of the transmembrane ephrin-B family ligandinduces trans-phosphorylation of the catalytically inactive EphB6receptor by its active EphB partner and brings the EphB6-Cb1 complexinto the proximity of the T cell receptor. The recruitment of EphB6 tothe immunological synapse downregulates activity of the TCR associatedkinases Lck and Zap-70, possibly by affecting cytoskeleton and TCRcomplex formation. This raises the threshold for T cell activation,which may serve to prevent T cell activation by low-affinity TCR-antigeninteraction. However, strong and sustained TCR stimulation causesphosphorylation of EphB6-associated Cb1, resulting in EphB6ubqiutination (Ub) and consequent downregulation. The removal of EphB6from the membrane allows complete activation o the TC signaling pathwayand subsequently, of T cell responses.

We claim:
 1. A method of modulating the immune system of an animalcomprising administering to the animal an effective amount of asubstance that modulates the expression, or activity of EphB6, or itsactive partner thereby modulating the immune system.
 2. A method ofmodulating of a cell comprising administering to the cell, an effectiveamount of a substance that modulates the expression, or activity ofEphB6, or its active partner thereby modulating the apoptosis.
 3. Amethod according to claim 1 or 2 wherein the substance is ephrin-B1, anoligomeric or monomeric soluble EphB6 receptor, a soluble EphB6 ligand,ephrin-B2, an antibody capable of binding EphB6, an antibody fragmentwhich is agonistic or antagonistic to EphB6, a physiological orsynthetic EphB6 ligand, a soluble active EphB6 partner, an antibody orfragments thereof to an EphB6 active partner, an antisense molecule toEphB6 or its active partners, or a physiological or synthetic ligand foran EphB6 active partner.
 4. A method according to claim 3 wherein thesubstance is Ephrin-B1 or Ephrin B2.
 5. A method of modulating cellproliferation comprising administering to the cell an effective amountof a substance which modulates the expression or activity of an EphB6receptor or its active partners.
 6. A method according to claim 5wherein the substance is ephrin-B1, an oligomeric or monomeric solubleEphB6 receptor, a soluble EphB6 ligand, ephrin-B2, an antibody capableof binding EphB6, an antibody fragment which is agonistic orantagonistic to EphB6, a physiological or synthetic EphB6 ligand, asoluble active EphB6 partner, an antibody or fragments thereof to anEphB6 active partner, an antisense molecule to EphB6 or its activepartners, or a physiological or synthetic ligand for an EphB6 activepartner.
 7. A method of modulating a T cell response in an animalcomprising administering to the animal an effective amount of asubstance that modulates EphB6 expression or activity or that of itspartner such that the T cell response is modulated.
 8. A methodaccording to claim 7 wherein the substance is ephrin-B1, an oligomericor monomeric soluble EphB6 receptor, a soluble EphB6 ligand, ephrin-B2,an antibody capable of binding EphB6, an antibody fragment which isagonistic or antagonistic to EphB6, a physiological or synthetic EphB6ligand, a soluble active EphB6 partner, an antibody or fragments thereofto an EphB6 active partner, an antisense molecule to EphB6 or its activepartners, or a physicological or synthetic ligand for an EphB6 activepartner.
 9. A method according to claim 8 wherein the substance isEphrin-B1 or Ephrin B2.
 10. A method according to anyone of claims 1-9wherein a substance which stimulates EphB6 is co-administered.
 11. Amethod according to claim 10 wherein the substance is ephrin B1, ephrinB2., or a catalytically active member of the EphB subfamily.
 12. Amethod according to claim 11 wherein the catalytically active member ofthe EphB subfamily is EphB1.
 13. A method of treating a disorder ofT-cell proliferation, an autoimmune disorder, a cell-associatedautoimmune disorder, an allergic disorder in an animal, or a host versustransplant reaction comprising administering to the animal an effectiveamount of a combination of inhibitory or stimulatory soluble EphB6ligand and/or soluble EphB6 receptor, or a ligand to an EphB6 activepartner or soluble partner, thereby treating the disorder.
 14. A methodaccording to claim 13 wherein the substance is ephrin-B1, an oligomericor monomeric soluble EphB6 receptor, a soluble EphB6 ligand, ephrin-B2,an antibody capable of binding EphB6, an antibody fragment which isagonistic or antagonistic to EphB6, a physiological or synthetic EphB6ligand, a soluble active EphB6 partner, an antibody or fragments thereofto an EphB6 active partner, an antisense molecule to EphB6 or its activepartners, or a physiological or synthetic ligand for an EphB6 activepartner.
 15. A method according to claim 13 wherein the cell-associatedautoimmunity is multiple sclerosis, lupus, arthritis, thyroiditis,diabetes, psoriasis, Crohn's disease or colitis.
 16. A method accordingto claim 13 wherein the allergic disorder is asthma, hyper-IgE syndrome,eosinophilic syndrome, or a T-cell dependent graft-verus-host disease.17. A method of promoting an anti-viral immune response in an animalcomprising administering to the animal an effective amount of asubstance that modulates the expression or activity of EphB6 or itsactive Eph partner thereby promoting the antiviral response in theanimal.
 18. A method according to claim 17 wherein the substance isephrin-B1, an oligomeric or monomeric soluble EphB6 receptor, a solubleEphB6 ligand, ephrin-B2, an antibody capable of binding EphB6, anantibody fragment which is agonistic or antagonistic to EphB6, aphysiological or synthetic EphB6 ligand, a soluble active EphB6 partner,an antibody or fragments thereof to an EphB6 active partner, anantisense molecule to EphB6 or its active partners, or a physiologicalor synthetic ligand for an EphB6 active partner.
 19. A method accordingto claim 17 wherein the substance is soluble stimulatory or inhibitoryephrin and/or a soluble EphB6 receptor.
 20. A method according to anyone of claims 1-17 wherein the animal is a human.
 21. A method foridentifying a substance which is capable of binding to a purified andisolated EphB6 protein, comprising reacting the protein with at leastone substance which potentially can bind with the protein underconditions which permit the formation of complexes between the substanceand the protein, and assaying for complexes, for free substance, fornon-complexed protein, or for activation of the protein.
 22. A methodfor assaying a medium for the presence of an agonist or antagonist ofthe interaction of a purified and isolated a EphB6 protein and asubstance which binds to the protein which comprises reacting theprotein with a substance which is capable of binding to the protein anda suspected agonist or antagonist substance under conditions whichpermit the formation of complexes between the substance and the protein,and assaying for complexes, for free substance, for non-complexedprotein, or for activation of the protein.